Patent Application
20240174722
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 15, 2022 is named 257723_000402_SL.txt and is 77,218 bytes in size.
The present disclosure relates generally to signal peptides and more particularly to synthetic signal peptides that increase secretion of a recombinant protein.
Yeasts are routinely used as hosts to produce proteins for research, therapeutic and industrial purposes. Once produced, a protein is usually translocated into the endoplasmic reticulum (ER), then transported to the Golgi, then secreted into the extracellular space. Movement along this secretory pathway is facilitated by a signal peptide which usually comprises about 16-30 amino acids and is fused to the N-terminus of the protein. However, despite considerable efforts to genetically optimize the synthesis of recombinant proteins by yeast, optimization of the chaperone pathways used by a synthesized protein to reach the extracellular space are comparatively fewer and have rarely been successful. The generation capacity of a yeast, therefore, remains too small to be viable for industrial-type applications and is thus limited to smaller scale processes.
The most common signal peptide used currently is the α-mating factor pro-protein signal peptide α-MF, from Saccharomyces cerevisiae. Its performance varies greatly depending on the payload protein. Only direct experimental assessment, with consequent expenditure of time and resources, provides assessment of its performance with any particular payload protein. Therefore, α-MF is usually implemented as is, not only in S. cerevisiae, but also in orthologous yeast strains, therefore compounding the unpredictability and challenge to effectively produce a recombinant protein in yeast. Some efforts to optimize secretion have been made but most, if not all have relied on either empirical design or directed evolution which are laborious and small scale method and require a native signal peptide as a starting template. A need therefore exists for engineering a system that not only increases the secretion of a recombinant protein produced in yeast, but has application across numerous yeast species.
In some embodiments, a pre-protein signal peptide is provided. In some embodiments, the pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII.
In certain embodiments, Formula I is represented by: A1-(A2)w-A3-(A4)x-(A5)y-A6-A7-A8-A9-A10-(A11)z (Formula I) as described herein.
In certain embodiments, Formula II is represented by: B1-(B2)u-(B3)v-(B4)w-(B5)x-(B6)y-B7-B8-B9-B10-(B11)z (Formula II) as described herein.
In certain embodiments, Formula III is represented by: C1-(C2)r-(C3)t-(C4)u-[(C5)v-(C6)w]x-(C7)y-(C8)z-C9-C10-C11-[C12-C13]a (Formula III) as described herein.
In certain embodiments, Formula IV is represented by: D1-(D2)q-(D3)r-(D4)t-(D5)u-[(D6)v-(D7)x-(D8)w-(D9)y]z-D10-D11-D12-[D13-D14]a (Formula IV) as described herein.
In certain embodiments, Formula V is represented by: E1-[(E2)i-(E3)j-(E4)q]r-(E5)t-(E6)u-(E7)v-[(E8)w-(E9)x]y-(E10)z-E11-E12-E13-[E14-E15]a (Formula V) as described herein.
In certain embodiments, Formula IX is represented by: F1-(F2)v-(F3)w-[(F4)x-(F5)y]z-F6-F7-F8-[F9-F10]a (Formula IX) as described herein.
In certain embodiments, Formula XIII is represented by: L1-(L2)x-[(L3)a-(L4)a]y-[(L5)a-(L6)a-(L7)a]z-(L8)a-(L9)a-(L10)a-(L11)a-(L12)a (Formula XIII) as described herein.
In some embodiments, a pre-protein signal peptide is provided. In some embodiments, the pre-protein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73.
In some embodiments, a pro-protein signal peptide is provided. In some embodiments, the pro-protein signal peptide comprises an amino acid sequence selected from the group consisting of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, and Formula XV.
In certain embodiments, Formula VI is represented by: G1-G2-G3-G4-G5-G6-G7-G8-G9-G10-G11-G12-G13-G14-G15-G16-G17-G18-G19-G20-G21-G22-G23-G24-G25 (Formula VI) as described herein.
In certain embodiments, Formula VII is represented by: (H1)m-(H2)m-(H3)m-(H4)m-(H5)m-(H6)m-(H7)m-(H8)m-(H9)m-(H10)m-(H11)m-(H12)m-(H13)m-(H14)m- (H15)m-(H16)m-(H17)m-(H18)m-(H19)m-(H20)m-(H21)m-(H22)m-(H23)m-(H24)m-(H25)m-(H26)m-(H27)m-(H28)m-(H29)m-(H30)m-(H31)m-(H32)m-(H33)m-(H34)m-(H35)m-(H36)m-H37-H38-H39-H40 (Formula VII) as described herein.
In certain embodiments, Formula VIII is represented by: (I1)m-(I2)m-(I3)m-(I4)m-(I5)m-(I6)m-(I7)x-(I8)m-(I9)m-(I10)m-(I11)x-(I12)m-(I13)x-(I14)x- (I15)m-(I16)x-(I17)m-I18-I19-I20-I21-I22-I23 (Formula VIII) as described herein.
In certain embodiments, Formula X is represented by: (J1)z-(J2)z-(J3)z-(J4)z-(J5)z-(J6)z-(J7)z-(J8)z-(J9)z-(J10)z-(J11)z-(J12)z-(J13)z-(J14)z-(J15)z- (J16)z-(J17)z-(J18)z-(J19)z-(J20)z-(J21)z-J22-J23-J24-J25 (Formula X) as described herein.
In certain embodiments, Formula XI is represented by: (K1)b-(K2)b-(K3)b-(K4)b-(K5)b-(K6)b-(K7)b-(K8)b-(K9)b-(K10)b-(K11)b-(K12)b-(K13)b-(K14)b- (K15)b-(K16)b-(K17)b-(K18)b-(K19)b-(K20)b-(K21)b-(K22)b-(K23)b-(K24)b-(K25)b-(K26)b-(K27)b-(K28)b-(K29)b-(K30)b-(K31)b-(K32)b-(K33)b-(K34)b-(K35)b-(K36)b-(K37)b-(K38)b-(K39)b-(K40)b-(K41)b-(K42)b-(K43)b-(K44)b-(K45)b-(K46)b-(K47)b-(K48)b-(K49)b-(K50)b- (K51)b-(K52)b-(K53)b-(K54)b-(K55)b-(K56)b-(K57)b-(K58)b-(K59)b-(K60)b-(K61)b-(K62)b-(K63)b-(K64)b-(K65)b-(K66)b-(K67)b-(K68)b-(K69)b-(K70)b-(K71)b-(K72)b-(K73)b-(K74)b-(K75)b-(K76)b-(K77)b-(K78)b-(K79)b-(K80)b-(K81)b-(K82)b-(K83)b-(K84)b-(K85)b-(K86)b-(K87)b-(K88)b-K89-K89-K89-K89-K89 (Formula XI) as described herein.
In certain embodiments, Formula XIV is represented by: (M1)b-(M2)b-(M3)b-(M4)b-(M5)b-(M6)b-(M7)b-(M8)b-(M9)b-(M10)b-(M11)b-(M12)b-(M13)b-(M14)b- (M15)b-(M16)b-(M17)b-(M18)b-(M19)b-(M20)b-(M21)b-(M22)b-(M23)b-(M24)b-(M25)b-(M26)b-(M26)b-(M27)b-(M28)b-(M29)b-(M30)b-(M31)b-(M32)b-(M33)b-(M34)b-(M35)b-(M36)b-(M37)b-(M38)b-(M39)b-(M40)b-(M41)b-(M42)b-(M43)b-(M44)b-(M45)b-(M46)b-(M47)b-(M48)b-(M49)b-(M50)b-(M51)b-(M52)b-(M53)b-(M54)b-(M55)b-(M56)b-(M57)b-(M58)b-(M59)b-(M60)b-(M61)b-(M62)b-(M63)b-(M64)b (M65)b-(M66)b-(M67)c-(M68)c-(M69)c-(M70)c (Formula XIV) as described herein
In certain embodiments, Formula XV is represented by: (N1)b-(N2)b-(N3)b-(N4)b-(N5)b-(N6)b-(N7)b-(N8)b-(N9)b-(N10)b-(N11)b-(N12)b-(N13)b-(N14)b- (N15)b-(N16)b-(N17)b-(N18)b-(N19)b-(N20)b-(N21)b-(N22)b-(N23)b-(N24)b-(N25)b-(N26)b-(N27)b-(N28)b-(N29)b-(N30)b-(N31)b-(N32)b-(N33)b-(N34)b-(N35)b-(N36)b-(N37)b-(N38)b-(N39)b-(N40)b-(N41)b-(N42)b-(N43)b-(N44)b-(N45)b-(N46)b-(N47)b-(N48)b-(N49)b-(N50)b-(N51)b-(N52)b-(N53)b-(N54)b-(N55)b-(N56)b-(N57)b-(N58)b-(N59)b-(N60)b-(N61)b-(N62)b-(N63)b-(N64)b-(N65)b-(N66)b-(N67)c-(N68)c-(N69)c-(N70)c-(N71)c (Formula XV) as described herein.
In some embodiments, a pro-protein signal peptide is provided. In some embodiments, the pro-protein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75.
In some embodiments, a pre-protein plus a pro-protein signal peptide is provided. In some embodiments, the pre-protein plus a pro-protein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence of SEQ ID NO: 30.
In some embodiments, a polypeptide is provided. In some embodiments, the recombinant polypeptide comprises a formula of (X1)n-(Y1)m-Z1, wherein X1 is a pre-protein signal peptide, Y1 is a pro-protein signal peptide, and Z1 is a payload protein, wherein n is 0 or 1 and m is 0 or 1, and wherein n and m cannot concurrently be 0.
In some embodiments, a yeast is provided. In some embodiments, the yeast comprises a heterologous nucleic acid molecule encoding a polypeptide having a formula of (X1)n-(Y1)m-Z1, wherein X1 is a pre-protein signal peptide as provided for herein, Y1 is a pro-protein signal peptide as provided for herein, and Z1 is a payload protein, wherein n is 0 or 1 and m is 0 or 1, and wherein n and m cannot concurrently be 0.
In some embodiments, a method for producing a payload protein is provided. In some embodiments, the method comprises transfecting a yeast with a nucleic acid encoding a recombinant polypeptide as provided for herein, producing an engineered yeast, culturing the engineered yeast in an environment effective to grow the engineered yeast, and inducing secretion of the payload protein by the engineered yeast.
In some embodiments, a method for treating a disease or condition in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a yeast as provided for herein.
The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.
The present disclosure presents a solution to the aforementioned challenges by providing new, synthetic signal peptides that direct secretion of expressed proteins or peptides in yeast. The disclosed signal peptides overcome performance variability challenges posed by previously characterized and native signal peptides and may be used to generate and facilitate secretion of any protein or peptide from a yeast.
The disclosed synthetic pre-protein (sPre) signal peptides and synthetic pro-protein (sPro) signal peptides increase secretion of any recombinant protein in yeast. Increased secretion can be advantageously achieved with a synthetic pre-protein signal peptide alone, with a synthetic pro-protein signal peptide alone, or with both. In any embodiment, a synthetic pre-protein signal peptide may be used in combination with a native pro-protein (nPro) signal peptide or sPro signal peptide. Likewise, in any embodiment, a synthetic pro-protein signal peptide may be used in combination with a native pre-protein (nPre) signal peptide or an sPre signal peptide. The use of synthetic pro-protein signal peptide together with a synthetic pre-protein signal peptide may further improve secretion of a payload protein, for example, through facilitating Golgi-trafficking. Advantageously, the signal peptides disclosed herein have been generated and optimized to promote secretion of any payload protein from a yeast. Use of the disclosed synthetic pre-protein signal peptides and synthetic pro-protein signal peptides may be used to achieve increased secretion of any desired payload to any yeast-compatible environment, such as in therapeutics, agriculture, or food products.
Before the present compositions and methods are described, it is to be understood that the scope of the invention is not limited to the particular processes, compositions, or methodologies described herein, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the methods and systems disclosed herein, the preferred methods, devices, and materials are now described.
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.
As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. For example, reference to “comprising a therapeutic agent” includes one or a plurality of such therapeutic agents. The term “or” refers to a single element of stated alternative elements, unless the context clearly indicates otherwise. For example, the phrase “A or B” refers to A alone or B alone. The phrase “A, B, or a combination thereof” refers to A alone, B alone, or a combination of A and B. Similarly, “one or more of A and B” refers to A, B, or a combination of both A and B. The phrase “A and B” refers to a combination of A and B. Furthermore, the various elements, features and steps discussed herein, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in particular examples.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. All references cited herein are incorporated by reference in their entirety.
In some examples, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments are to be understood as being modified in some instances by the term “about” or “approximately.” For example, “about” or “approximately” can indicate +/−5% variation of the value it describes. Accordingly, in some embodiments, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties for a particular embodiment. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some examples are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.
To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:
As used herein, “yeast” refers to a microscopic fungus consisting of cells that reproduce by budding and are capable of converting sugar into alcohol and carbon dioxide. The yeast, as disclosed herein may be genetically modified to induce expression of a heterologous payload protein. As used herein, “genetically modified” or any grammatical variation thereof, refers to a practice of introducing a nucleic acid or a nucleic acid molecule into a yeast cell that encodes and promotes the expression of a recombinant protein. The nucleic acid may be introduced transiently, or the nucleic acid may be incorporated into the genome of the yeast for stable expression. As used herein, the terms “nucleic acid” and “nucleic acid molecule” can be used interchangeably. The nucleic acid or nucleic acid molecule can be of any length. A nucleic acid may be DNA, mRNA, tRNA, or rRNA. A nucleic acid or nucleic acid molecule is composed of nucleotide monomers, each triplet of monomers (a codon) encoding for either a triplet of RNA nucleotide monomers (if the nucleic acid is DNA) or an amino acid (if the nucleic acid is RNA). DNA also comprises one or more promoter regions, which indicate where transcription of the DNA should start. mRNA also comprises a ribosome binding site, which indicates where translation of the mRNA should start as well as one or more stop codons, which indicates where mRNA translation should end. The introduction of a nucleic acid or nucleic acid molecule into a yeast cell can be accomplished by any method known in the art. Such methods are described in greater detail below.
In any embodiment or aspect disclosed herein, a nucleic acid encoding for a recombinant polypeptide, as disclosed herein, may be introduced into a yeast cell using any method known to those skilled in the art for such introduction. Such methods include transfection, transformation, transduction, infection (e.g., viral transduction), injection, microinjection, gene gun, nucleofection, nanoparticle bombardment, transformation, conjugation, by application of the nucleic acid in a gel, oil, or cream, by electroporation, using lipid-based transfection reagents, or by any other suitable transfection method. One of skill in the art will readily understand and adapt such methods using readily identifiable literature sources.
As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as, for example, LIPOFECTIN® (Invitrogen Corp., San Diego, CA), LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, NY), EFFECTENE® (Qiagen, Valencia, CA), DREAMFECT™ (OZ Biosciences, France) and the like), or electroporation (e.g., in vivo electroporation). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
Methods and materials of non-viral delivery of nucleic acids to cells further include biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid-nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355 and lipofection reagents are sold commercially (e.g., TRANSFECTAM™ and LIPOFECTIN™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO91/17424 and WO 91/16024.
The methods described herein comprise generating a recombinant polypeptide within a yeast host. As used herein, heterologous or recombinant describes a protein or nucleic acid that is not naturally found in or produced by the host yeast. As used herein, a “recombinant polypeptide” comprises a payload protein and a synthetic signal peptide fused directly or indirectly thereto. As used herein, “recombinant polypeptide” and “recombinant fusion protein” may be used interchangeably in the context of polypeptides comprising at least a first and second component (e.g. a synthetic signal peptide and a payload protein). As used herein, a signal peptide is any protein or peptide fused directly or indirectly to the N-terminus of a payload protein that facilitates the extracellular secretion of the payload protein after it is generated. A signal peptide may comprise one or more of a pre-protein signal peptide and pro-protein signal peptide.
While not wishing to be bound by theory, it is thought that the synthetic pre-protein signal peptides disclosed herein facilitate efficient translocation of the protein from a ribosome to the endoplasmic reticulum, and that the synthetic pro-protein signal peptides disclosed herein facilitate trafficking of the protein from the ER to the Golgi apparatus for eventual secretion. Pro-protein signal peptides are known to regulate a different types of cellular processes, such as transport and localization, hierarchical organization and oligomerization, including facilitation of proper protein folding, and regulation of protein activity-function. Further, inclusion of a pro-protein signal peptide can enrich for the amount of protein in certain cellular localizations. For example, inclusion of a pro-protein sequence peptide on a protein of interest can enrich for the amount of the protein of interest in the paraplasm of yeast. In the context of facilitating translocation, the effect of the pre-protein signal peptide, pro-protein signal peptide, or combination thereof as described herein is target dependent. While not wishing to be bound by theory, in some embodiments a pre-protein signal peptide without the pro-protein signal peptide will facilitate more efficient translocation and secretion. In some embodiments, a pro-protein signal peptide without the pre-protein signal peptide will facilitate more efficient translocation and secretion. In some embodiments, inclusion of both the pre and pro-protein signal peptides will facilitate more efficient secretion.
The chemical makeup of a peptide will be described herein by a series of amino acid single letter abbreviations or an “amino acid sequence/s” or “sequence/s,” which are conventional and known to those in the art. While reference sequences will be explicitly disclosed, in any aspect and embodiment, a reference sequence may be modified to include conservative amino acid substitutions, as well as variants and fragments, while maintaining the characteristics and functionality of the reference sequence.
The methods disclosed herein utilize a synthetic signal peptide to increase extracellular secretion of a payload protein by a yeast. As used herein, a “synthetic signal peptide” refers to a signal peptide whose sequence is generated as provided for herein and that is made recombinantly. The recombinantly produced signal peptide can be referred to as a “synthetic signal peptide” or simply as a “signal peptide”. The signal peptide comprising one or more of a synthetic pre-protein (sPre) signal peptide and a synthetic pro-protein (sPro) signal peptide. As highlighted previously, the term synthetic in this context refers to a recombinantly produced pre-protein signal peptide or pro-protein signal peptide whose sequence is generated as provided for herein. Hereafter, the pre- and pro-signal peptides may be referred to as “synthetic” pre or pro-protein signal peptides, or simply as pre or pro-protein signal peptides. In embodiments where a native pre or pre-protein signal peptide is utilized or referred to, the peptide will be denoted as such. In the context of this application, the term “native” refers to a pre or pro signal peptide the sequence of which is adopted, in whole or in part, from a known pre or pro signal peptide sequence at the time of this application. In other words, the “native” signal peptides are not generated using the formulas or methods as provided for herein. However, it is to be understood that a synthetic signal peptide may comprise a synthetic pre-protein signal peptide fused with a native pro-protein signal peptide (sPre-nPro signal peptide). In another example, a synthetic signal peptide may comprise a native pre-protein signal peptide fused to a synthetic pro-protein signal peptide (nPre-sPro signal peptide). In yet another example, a synthetic signal peptide comprises a synthetic pre-protein signal peptide and no pro-protein signal peptide. Similarly, a synthetic signal peptide may comprise a synthetic pro-protein signal peptide but no pre-protein signal peptide.
A pre-protein signal peptide (synthetic or native) comprises 10 to 50 amino acids, which are appended either directly to the N-terminus of a payload protein or indirectly to the N-terminus of a payload protein, with one or more of a Kex protease (KR) site, Ste13 cleavage site, and spacer there between.
A pro-protein signal peptide comprises 10 to 200 amino acids that are appended either directly to the N-terminus of a payload protein or indirectly to the N-terminus of a payload protein, with one or more of a KR site, Ste13 cleavage site, and spacer there between. Many proteins are natively expressed comprising a pro-protein signal peptide, though, as will be described, these native pro-protein signal peptides often lack the activity to generate sufficient secretion of a payload protein. The various synthetic signal peptides described herein may be used as a replacement of all or part of a native signal peptides.
A pre- and/or pro-protein signal peptide, whether synthetic or native, may be appended to an adjacent amino acid via a bond to the N-terminal amino acid of the adjacent amino acid, for example, by a peptide bond, a dipeptide spacer, or a membrane-associating/lipidophilic alpha-helical peptide signal peptide (e.g., MISTIC, represented by the amino acid sequence
As used herein, “hydropathy index” or “HP index” refers to the “intrinsic” hydrophobicity/hydrophilicity of amino acid side chains in peptides/proteins as defined in Kovacs J M, Mant C T, Hodges R S. Determination of intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides in the absence of nearest-neighbor or conformational effects. Biopolymers. 2006; 84(3):283-97. doi: 10.1002/bip.20417. PMID: 16315143; PMCID: PMC2744689, which is hereby incorporated by reference in its entirety. Hydrophobicity/hydrophilicity values were determined via a synthetic peptide wherein the HP index value is calculated as the difference in RP-HPLC retention time between amino acid X at the i position and amino acid Gly at the i+1 position. Thus, amino acids that are more hydrophobic than glycine have a positive HP index value and amino acids that are more hydrophilic than glycine have a negative HP index value, wherein glycine would have a 0 value. See Table 1 below, values which correspond to the values utilized for the present application.
As used herein “helicity” refers to the nonpolar phase helical propensity of each guest “X” residue in an experimental KKAAAXAAAAAXAAWAAXAAAKKKK (SEQ ID NO. 84)—amide peptide, as outlined in Deber C M, Wang C, Liu L P, Prior A S, Agrawal S, Muskat B L, Cuticchia A J. TM Finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales. Protein Sci. 2001 January; 10(1):212-9. doi: 10.1110/ps.30301. PMID: 11266608; PMCID: PMC2249854, which is hereby incorporated by reference in its entirety. Helicity values for each amino acid are in Table 2 below.
As used herein, “payload protein” or “protein of interest” refers to the protein that will be generated by the host and chaperoned through the secretory pathway into the extracellular space, facilitated by the presence of a synthetic signal peptide. Upon secretion into the extracellular space, all, some, or none of the synthetic signal peptide may be fused to the payload protein. Optionally, a payload protein still being attached partially or fully to the synthetic signal peptide may be further processed, for example, to remove the remaining signal peptide. A payload protein may be any protein known or yet to be known, for example, an enzyme, enzyme inhibitor, growth factor, hormone, antibody, antigen, vaccine, a therapeutic agent, or any combination thereof. More specific examples follow herein below.
The compositions disclosed herein may be provided to a subject in a variety of ways through administration of the composition to the subject. As used herein, administer or administration means to provide or the providing of a composition to a subject. Oral administration, as used herein, refers to delivery of an active agent through the mouth. Topical administration, as used herein, refers to the delivery of an active agent to a body surface, such as the skin, a mucosal membrane (e.g., nasal membrane, vaginal membrane, buccal membrane, or the like).
A payload protein secreted by the various genetically modified yeast disclosed herein, which are interchangeably referred to as “engineered yeast”, may be provided to a subject in a pharmaceutical composition. Additionally or alternatively, the engineered yeast itself may be provided to a subject in a pharmaceutical composition.
The various compositions disclosed herein may be useful in treating a number of diseases, for example, cancer. As used herein, cancer refers to a condition characterized by unregulated cell growth. Examples of cancer include, but are not limited to, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervical cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, and esophageal cancer. In some embodiments, the diseases or conditions may include, but is not limited to, an infection, an autoimmune disease, enzymatic deficiencies (including primary (congenital) enzymatic deficiency and enzymatic deficiencies secondary to functional gut disorders), diabetes, obesity, metabolic disorders, intestinal bacterial overgrowth, enteric infection, bacterial vaginosis, short bowel syndrome, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, Celiac disease, gluten intolerance, colitis, peptic ulcer, gastritis, polyps, hemorrhoids, cirrhosis, or a cancer
The various compositions disclosed herein may comprise one or more drugs, biologics, or active agents, which are used interchangeably herein and refer to a chemical substance or compound that induces a desired pharmacological or physiological effect, and includes agents that are therapeutically effective, prophylactically effective, or cosmetically effective. “Drug,” “biologic,” and “active agent” include any pharmaceutically acceptable, pharmacologically active derivatives and analogs of those drugs, biologics, and active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, inclusion complexes, analogs, and the like. Suitable drugs, biologics, and active agents may include, but are not limited to, alcohol deterrents; amino acids; ammonia detoxicants; anabolic agents; analeptic agents; analgesic agents; androgenic agents; anesthetic agents; anorectic compounds; anorexic agents; antagonists; anti-allergic agents; anti-amebic agents; anti-anemic agents; anti-anginal agents; anti-anxiety agents; anti-arthritic agents; anti-atherosclerotic agents; anti-bacterial agents; anti-cancer agents, including antineoplastic drugs, and anti-cancer supplementary potentiating agents; anticholinergics; anticholelithogenic agents; anti-coagulants; anti-coccidal agents; anti-convulsants; anti-depressants; anti-diabetic agents; anti-diarrheals; anti-diuretics; antidotes; anti-dyskinetics agents; anti-emetic agents; anti-epileptic agents; anti-estrogen agents; anti-fibrinolytic agents; anti-fungal agents; anti-glaucoma agents; anti-hemophilic agents; anti-hemorrhagic agents; antihistamines; anti-hyperlipidemic agents; anti-hyperlipoproteinemic agents; antihypertensive agents; anti-hypotensives; anti-infective agents such as antibiotics and antiviral agents; anti-inflammatory agents, both steroidal and non-steroidal; anti-keratinizing agents; anti-malarial agents; antimicrobial agents; anti-migraine agents; anti-mitotic agents; anti-mycotic agents; antinauseants; antineoplastic agents; anti-neutropenic agents; anti-obsessional agents; anti-parasitic agents; antiparkinsonism drugs; anti-pneumocystic agents; anti-proliferative agents; anti-prostatic hypertrophy drugs; anti-protozoal agents; antipruritics; anti-psoriatic agents; antipsychotics; antipyretics; antispasmodics; anti-rheumatic agents; anti-schistosomal agents; anti-seborrheic agents; anti-spasmodic agents; anti-thrombotic agents; anti-tubercular agents; antitussive agents; anti-ulcerative agents; anti-urolithic agents; antiviral agents; GERD medications, anxiolytics; appetite suppressants; attention deficit disorder (ADD) and attention deficit hyperactivity disorder (ADHD) drugs; bacteriostatic and bactericidal agents; benign prostatic hyperplasia therapy agents; blood glucose regulators; bone resorption inhibitors; bronchodilators; carbonic anhydrase inhibitors; cardiovascular preparations including anti-anginal agents, anti-arrhythmic agents, beta-blockers, calcium channel blockers, cardiac depressants, cardiovascular agents, cardioprotectants, and cardiotonic agents; central nervous system (CNS) agents; central nervous system stimulants; choleretic agents; cholinergic agents; cholinergic agonists; cholinesterase deactivators; coccidiostat agents; cognition adjuvants and cognition enhancers; cough and cold preparations, including decongestants; depressants; diagnostic aids; diuretics; dopaminergic agents; ectoparasiticides; emetic agents; enzymes which inhibit the formation of plaque, calculus or dental caries; enzyme inhibitors; estrogens; fibrinolytic agents; fluoride anticavity/antidecay agents; free oxygen radical scavengers; gastrointestinal motility agents; genetic materials; glucocorticoids; gonad-stimulating principles; hemostatic agents; herbal remedies; histamine H2 receptor antagonists; hormones; hormonolytics; hypnotics; hypocholesterolemic agents; hypoglycemic agents; hypolipidemic agents; hypotensive agents; immunizing agents; immunomodulators; immunoregulators; immunostimulants; immunosuppressants; impotence therapy adjuncts; inhibitors; keratolytic agents; leukotriene inhibitors; liver disorder treatments; metal chelators such as ethylenediaminetetraacetic acid, tetrasodium salt; mitotic inhibitors; mood regulators; mucolytics; mucosal protective agents; muscle relaxants; mydriatic agents; narcotic antagonists; neuroleptic agents; neuromuscular blocking agents; neuroprotective agents; nicotine; NMDA antagonists; non-hormonal sterol derivatives; nutritional agents, such as vitamins, essential amino acids and fatty acids; ophthalmic drugs such as antiglaucoma agents; oxytocic agents; pain relieving agents; parasympatholytics; peptide drugs; plasminogen activators; platelet activating factor antagonists; platelet aggregation inhibitors; post-stroke and post-head trauma treatments; potentiators; progestins; prostaglandins; prostate growth inhibitors; proteolytic enzymes as wound cleansing agents; prothyrotropin agents; psychostimulants; psychotropic agents; radioactive agents; regulators; relaxants; repartitioning agents; scabicides; sclerosing agents; sedatives; sedative-hypnotic agents; selective adenosine A1 antagonists; serotonin antagonists; serotonin inhibitors; serotonin receptor antagonists; steroids, including progestogens, estrogens, corticosteroids, androgens and anabolic agents; smoking cessation agents; stimulants; suppressants; sympathomimetics; synergists; thyroid hormones; thyroid inhibitors; thyromimetic agents; tranquilizers; tooth desensitizing agents; tooth whitening agents such as peroxides, metal chlorites, perborates, percarbonates, peroxyacids, and combinations thereof; unstable angina agents; uricosuric agents; vasoconstrictors; vasodilators including general coronary, peripheral and cerebral; vulnerary agents; wound healing agents; xanthine oxidase inhibitors; and the like.
Antibiotic refers to a chemical substance capable of treating bacterial infections by inhibiting the growth of, or by destroying existing colonies of bacteria and other microorganisms.
Anti-inflammatory refers to an active agent that reduces inflammation and swelling.
Chemotherapeutic agent refers to a chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer. In one example, a chemotherapeutic agent is a radioactive compound. In one example, a chemotherapeutic agent is a biologic, such as a monoclonal antibody. Chemotherapy refers to use of a chemotherapeutic agent.
Radiation therapy refers to use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether.
The various compositions disclosed herein may comprise an effective amount of a drug, biologic, or active agent. Effective amount refers to an amount of a drug, biologic, or active agent (alone or with one or more other active agents) sufficient to induce a desired response, such as to prevent, treat, reduce and/or ameliorate a condition. An effective amount of an active agent, alone or with one or more other active agents, can be determined in many different ways, such as assaying for a reduction in of one or more signs or symptoms associated with the condition in the subject or measuring the level of one or more molecules associated with the condition to be treated.
The various compositions disclosed herein may comprise various pharmaceutically acceptable excipients. As used herein, a pH adjuster or modifier refers to a compound or buffer used to achieve desired pH control in a formulation. Exemplary pH modifiers include acids (e.g., acetic acid, adipic acid, carbonic acid, citric acid, fumaric acid, phosphoric acid, sorbic acid, succinic acid, tartaric acid), bases (e.g., magnesium oxide, tribasic potassium phosphate), and pharmaceutically acceptable salts thereof.
Pharmaceutically acceptable carriers useful in this disclosure are those conventionally known in the art. The nature of the carrier can depend on the particular mode of administration being employed. For instance, oral applications usually include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like, as a vehicle. In addition to biologically-neutral carriers, oral compositions may also contain auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, and the like.
Antioxidant refers to a compound that inhibits oxidation or reactions promoted by oxygen or peroxides.
Mucoadhesive refers to a substance that strongly attaches to mucosa upon hydration without any additional adhesive material, and remains adhered to the tissue in vivo.
In some embodiments, synthetic signal peptides that increase secretion of a payload protein from yeast are provided. In some embodiments, the synthetic signal peptide, as described above, comprises one or more of a synthetic pre-protein signal peptide and pro-protein signal peptide. In any embodiment, a native pre- or pro-protein signal peptide may be combined with a synthetic signal peptide, provided at least one of the pre- and pro-protein signal peptide is synthetic. In some embodiments, recombinant polypeptides are provided comprising a synthetic signal peptide and a payload protein, wherein the synthetic signal peptide is fused, either directly or indirectly, to the payload protein. In some embodiments, the synthetic signal peptide is fused directly to the protein of interest. In some embodiments, the synthetic signal peptide and protein of interest are connected via a peptide linker. Suitable peptide linkers are known in the art and any such linker may be utilized. In some embodiments, the linker is a flexible peptide linker. In some embodiments, the linker is a non-cleavable peptide linker. In some embodiments the linker is a cleavable peptide linker. In some embodiments, the recombinant polypeptide comprises a synthetic pre-protein signal peptide and a payload protein. For example,
Table 3 below lists various amino acid sequences that will be referred to herein. In Table 3, amino acids contained within parentheses are optional. It is to be understood that when multiple amino acids are contained within parentheses, any one of the amino acids can be added or excluded without the addition of the other. The sequences EEGEPK (SEQ ID NO. 78) and DVVYPK (SEQ ID NO. 79) are spacers and DKREEGPK (SEQ ID NO. 80), KREEGPK (SEQ ID NO. 81), DKREKRE (SEQ ID NO. 82), and DKR (SEQ ID NO. 83) are Kex protease sites.
cerevisiae)
cerevisiae)
lactis)
lactis)
In addition to the Kex protease sites recited in the above examples, the pre-protein signal peptides and pro-protein signal peptides of the present disclosure may also optionally contain a KEX2 cleavage site, as given by the amino acid sequence NVISKR (SEQ ID NO. 68), or the amino acid sequence SDVTKR (SEQ ID NO. 69). In any embodiment, the sequence of SEQ ID NO. 68 can be appended to the C-terminus or N-terminus of any pre- or pro-protein signal peptide as provided for herein. Accordingly, in some embodiments, the pre-protein signal peptide is as provided. In some embodiments, the pro-protein signal peptide is as provided. In any embodiment, the sequence of SEQ ID NO. 69 can be appended to the C-terminus or N-terminus of any pre- or pro-protein signal peptide as provided for herein. Accordingly, in some embodiments, the pre-protein signal peptide is as provided. In some embodiments, the pro-protein signal peptide is as provided.
In some embodiments, the KEX2 cleavage site can be represented by the following formula:
X4X3X2X1B1B2 (Formula XII)
wherein i) X1, X2, and X3 are not G, ii) X1 is not 5, if X2 and X3 are G, X4 is A, or X5 is 5, iii) X4 is not T, if X3 is A and X2 is 5; or iv) X1 is not D; and wherein B1 and B2 are each, independently, basic amino acids. The details of Formula XII are described in U.S. Pat. No. 8,936,917, which is hereby incorporated by reference in its entirety. Accordingly, in any embodiment, the sequence of Formula XII can be appended to the C-terminus or N-terminus of any pre- or pro-protein signal peptide as provided for herein. In some embodiments, the pre-protein signal peptide is as provided. In some embodiments, the pro-protein signal peptide is as provided.
Any synthetic pre-protein or pro-protein signal peptide may be combined with some or all of a known signal peptide. Examples of known signal peptides that may be combined with any of SEQ ID Nos 1-25, 31-38, 55-58, and 70-75 in Table 3 to generate a synthetic signal peptide include, but are not limited to, HSp150, PH05, SUC2, KILM1, GGP1, SUN, PLB, CRH, EXG, AGA2, HAS pre-pro, PIR1, XPR2 pre, XPR2 pre-pro, pGKL, SCW, and DSE.
One who is skilled in the art will be able to develop a nucleic acid that encodes for the expression of any one of SEQ ID NOs. 1-38, 55-58, and 70-75. Table 4 below provides example nucleotide sequences that may be used to generate the synthetic peptides described in Table 3. It is to be understood that the nucleic acid sequences provided in Table 4 are exemplary and are not meant to be limiting in any way. Due to the degenerate nature of codons, other nucleic acid molecules can be used. In some embodiments, the nucleic acid molecule is codon optimized for expression in a bacterial system. In some embodiments, the nucleic acid molecule is codon optimized for expression in a eukaryotic system or cell.
The synthetic signal peptides disclosed herein are optimized for use in yeast and can be used to induce expression of any protein. Particular examples of suitable yeast species are provided herein below to exemplify the particular synthetic signal peptides that have been developed.
As noted above, Table 3 discloses amino acid sequences, however, in any aspect and embodiment, any of the sequences in Table 3 may be modified with conservative amino acid substitutions to produce active variants that maintain the characteristics and functionality of the primary sequence. These conservative amino acid substitutions can be generally described by the Formulas below, which encapsulate the consensus sequence as well as the variant sequences. The various Formulas detailing the variant sequences will now be described.
In some embodiments, a pre-protein signal peptide is provided. In some embodiments, the pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII.
In some embodiments, the pre-protein signal peptide comprises an amino acid sequence represented by:
A1-(A2)w-A3-(A4)x-(A5)y-(A6)-(A7)-(A8)-(A9)-(A10)-(A11)z (Formula I)
wherein:
In some embodiments, w is 1. In some embodiments w is 2. In some embodiments, w is 3. In some embodiments, w is 4. In some embodiments, w is 5. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 5. In some embodiments, y may be an integer selected from 2-18, 4-16, 6-14, 8-12, 7-11, and 8-10. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. It is to be understood that the values of w, x, y, and z are each independently selected, and the value of any variable w, x, y, or z is independent of the values selected for the other variables. In some embodiments, each A3, A5, A8, and A10 is each, independently, an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments, each A3, A5, A8, and A10 is each, independently, an amino acid selected from the group consisting of L, V, A, and I. In some embodiments A3 is each an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments, A3 is an amino acid selected from the group consisting of L, V, A, and I. In some embodiments A5 is each an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments, A5 is an amino acid selected from the group consisting of L, V, A, and I. In some embodiments A8 is each an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments A8 is an amino acid selected from the group consisting of L, V, A, and I. In some embodiments A10 is each an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments A10 is an amino acid selected from the group consisting of L, V, A, and I. In some embodiments, each A11 is, independently, an amino acid selected from the group consisting of N, S, T, C, A, V, G, I, L, and P. In some embodiments, each A11 is, independently, an amino acid selected from the group consisting of A, L, and G. In some embodiments, each A2 is, independently, an amino acid selected from the group consisting of K, R, H and Q. In embodiments where any one of w, x, y, and z are an integer greater than 1, each amino acid in the group described by the w, x, y, and z are independently chosen from the disclosed group of amino acids and therefore may be the same or different. For example, for (A2)w wherein w is 3, this grouping expands to A2A2A2 where each A2 is, independently, a neutral or positively-charged amino acid with a hydropathy index less than about 1. This meaning, unless explicitly indicated otherwise, expands to all further formulas disclosed herein and below.
In some embodiments, the sequence of SEQ ID NO. 1 can be derived from Formula I as follows: w is 1, x is 2, y is 9, and z is 2; A1 is methionine; A2 is K; A3 is L; both the first and second instances of A4 are S; all 9 instances of A5 are L; A6 is S; A7 is S; A8 is L; A9 is V; A10 is L; and both instances of A11 are A.
In certain embodiments, the pre-protein signal peptide comprises an amino acid sequence represented by:
B1-(B2)u-(B3)v-(B4)w-(B5)x-(B6)y-(B7)-(B8)-(B9)-(B10)-(B11)z (Formula II)
wherein:
In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments x is 0. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, y may be an integer selected from 2-18, 4-16, 6-14, 8-12, 7-11, and 8-10. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. It is to be understood that the values of u, w, v, z, x, and y are each independently selected, and the value of any variable u, w, v, z, x, or y is independent of the values selected for the other variables. In some embodiments, each B2, B4, B6, B8, and B10 is each, independently, an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, each B2, B4, B6, B8 and B10 is each, independently, an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, each B2 is, independently, an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, each B2 is, independently, an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, each B4 is, independently, an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, each B4 is, independently, an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, each B6 is, independently, an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, each B6 is, independently, an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, B8 is an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, B8 is an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, B10 is an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, B10 is an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, each B5 is, independently, an amino acid selected from the group consisting of K, R, E, D, G, A, V, L, I, F, S, T, Y, N, and H. In some embodiments, each B5 is, independently, an amino acid selected from the group consisting of K, R, E, and D. In some embodiments, each B5 is, independently, an amino acid selected from the group consisting of G, A, V, L, I, F, S, T, Y, N, K, R, and H. In some embodiments, each B7 and B11 is each, independently, an amino acid selected from the group consisting of A, S, G, and P. In some embodiments, B7 is an amino acid selected from the group consisting of A, S, G, and P. In some embodiments, each B11 is, independently, an amino acid selected from the group consisting of A, S, G, and P. In some embodiments, B9 is an amino acid selected from the group consisting of A, C, G, I, L, M, F, S, T, W, Y, V, N, Q, D, E, and P. In some embodiments, each B3 is each, independently, an amino acid selected from the group consisting of K, R, H and Q. In embodiments where any one of u, w, v, z, x and y are an integer greater than 1, each amino acid in the group described by the u, w, v, z, x and y are independently chosen from the disclosed group of amino acids and therefore may be the same or different, as described for herein.
In some embodiments, the sequence of SEQ ID NO. 4 can be derived from Formula II as follows: u is 0, v is 1, w is 1, x is 1, y is 11, and z is 3; B1 is methionine; B2 is absent; B3 is K; B4 is L; B5 is S; the string of eleven (11) B6 residues is as follows: T-L-L-L-T-L-L-L-L-L-L (SEQ ID NO: 87); B7 is A; B8 is L; B9 is V; B10 is L; and the string of three (3) B11 residues is as follows: A-A-S.
In some embodiments, the sequence of SEQ ID NO. 5 can be derived from Formula II as follows: u is 1, v is 1, w is 1, x is 0, y is 11, and z is 3; B1 is methionine; B2 is L; B3 is K; B4 is L; B5 is absent; the string of eleven (11) B6 residues is as follows: L-L-L-I-L-L-L-L-L-L-V (SEQ ID NO: 88); B7 is S; B8 is L; B9 is V; B10 is L; and the string of three (3) B11 residues is as follows: A-A-S.
In some embodiments, the sequence of SEQ ID NO. 6 can be derived from Formula II as follows: u is 0, v is 1, w is 0, x is 0, y is 15, and z is 3; B1 is methionine; B2 is absent; B3 is K; B4 is absent; B5 is absent; all fifteen (15) B6 residues are L; B7 is A; B8 is L; B9 is V; B10 is L; and the string of three (3) B11 residues is as follows: A-A-S.
In some embodiments, the sequence of SEQ ID NO. 7 can be derived from Formula II as follows: u is 0, v is 1, w is 0, x is 0, y is 6, and z is 3; B1 is methionine; B2 is absent; B3 is K; B4 is absent; B5 is absent; all six (6) B6 residues are L; B7 is S; B8 is L; B9 is V; B10 is L; and the string of three (3) B11 residues is as follows: A-A-S.
In some embodiments, the pre-protein signal peptide comprises an amino acid sequence represented by:
C1-(C2)r-(C3)t-(C4)u-[(C5)v-(C6)w]x-(C7)y-(C8)z-(C9)-(C10)-(C11)-[(C12)-(C13)]a (Formula III)
wherein C2-C13 have the properties described in Table 5 below:
−4-34
wherein r is an integer selected from 1-3;
In some embodiments, C1 is methionine. In some embodiments, each C2 is, independently, an amino acid having an isoelectric point of about 5.6 to about 10.8, a molecular weight of about 105 g/mol to about 175 g/mol, a hydropathy index of about −5.1 to about 0.6, and a helicity of about 0.8 to about 1. In some embodiments, each C3, C5, C8, and C10 is each, independently, an amino acid having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each C3 is, independently, an amino acid having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each C5 is, independently, an amino acid having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each C8 is, independently, an amino acid having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, C10 is an amino acid having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each C4 and C7 is each, independently, an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each C4 is, independently, an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each C7 is, independently, an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each C6, C9, C11, and C12 is each, independently, an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −4 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each C6 is each, independently, an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −4 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, C9 is an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −4 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, C11 is an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −4 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, C12 is an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −4 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, C13 is an amino acid having an isoelectric point of about 5.6 to about 6.3, a molecular weight of about 105 g/mol to about 120 g/mol, a hydropathy index of about 0 to about 9.4, and a helicity of about 0.5 to about 1.1.
In some embodiments, r is 1. In some embodiments, r is 2, in some embodiments, r is 3. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, y is 0. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, z is 0. In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, x may be an integer selected from 3-9, 4-8, 6-10, 8-10, 2-5, and 3-6. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 5. In some embodiments, x is 6. In some embodiments, x is 7. In some embodiments, x is 8. In some embodiments, x is 9. In some embodiments, x is 10. In some embodiments a is 0 and the residues given by [(C12)-(C13)]a are absent. In some embodiments, a is 1 and the residues given by [(C12)-(C13)]a are present. It is to be understood that the values of r, t, u, y, z, v, w, and x are each independently selected, and the value of any variable r, t, u, y, z, v, w, or x is independent of the values selected for the other variables. In some embodiments, each C3, C5, C8, and C10 is each independently, an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, each C3, C5, C8, and C10 is each, independently, an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, each C3 is, independently, an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, each C3 is, independently, an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, each C5 is, independently, an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, each C5 is, independently, an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, each C8 is, independently, an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, each C8 is, independently, an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, C10 is an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, C10 is an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, each C6, C9, C11, and C12 is each, independently, an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, each C6, C9, C11, and C12 is each, independently, an amino acid selected from the group consisting of A and S. In some embodiments, each C6 is, independently, an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, each C6 is, independently, an amino acid selected from the group consisting of A and S. In some embodiments, C9 is an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, C9 is an amino acid selected from the group consisting of A and S. In some embodiments, C11 is an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, C11 is an amino acid selected from the group consisting of A and S. In some embodiments, C12 is an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, C12 is an amino acid selected from the group consisting of A and S. In some embodiments, each C2 is, independently, an amino acid selected from the group consisting of K, R, H, S, and Q. In some embodiments, C13 is an amino acid selected from the group consisting of P, T, and S. In some embodiments, each C4 and C7 is each, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, A, Y, H, V, I, F, G, W, C, P, and L. In some embodiments, each C4 and C7 is each, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, A, and Y. In some embodiments, each C4 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, A, Y, H, V, I, F, G, W, C, P, and L. In some embodiments, each C4 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, A, and Y. In some embodiments, each C7 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, A, Y, H, V, I, F, G, W, C, P, and L. In some embodiments, each C7 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, A, and Y. In embodiments where any one of r, t, u, y, z, v, w, and x are an integer greater than 1, each amino acid in the group described by the r, t, u, y, z, v, w, and x are independently chosen from the disclosed group of amino acids and therefore may be the same or different, as described for herein.
Further, in consideration of [(C5)v-(C6)w]x, it is to be understood that in embodiments where x is an integer greater than 1, the formula [(C5)v-(C6)w]x does not indicate that [(C5)v-(C6)w] is repeated x number of times. Rather, when (C5)v-(C6)w is expanded due to an x greater than 1, each instance of C5 can independently be selected from an appropriate amino acid as detailed above and likewise each instance of C6 can independently be selected from an appropriate amino acid as detailed above. For example, in considering a hypothetical of [(C5)1-(C6)1]2, the formula could produce the sequence L-A-L-A (SEQ ID NO: 101) wherein the first and second C5 are both L and the first and second C6 are both A, and could likewise produce L-A-V-C(SEQ ID NO: 102), wherein the first C5 is L, the first C6 is A, the second C5 is V, and the second C6 is C. In a further example, in considering a hypothetical of [(C5)1-(C6)1]3, the formula could produce the sequence L-A-L-A-L-A (SEQ ID NO: 103) wherein the first, second, and third C5 are all L and the first, second, and third C6 are all A, and could likewise produce L-A-V-C-H-P (SEQ ID NO: 104), wherein the first C5 is L, the first C6 is A, the second C5 is V, the second C6 is C, the third C5 is H, and the third C6 is P. The same functionality of x holds true for the values of v and w. For example, in considering a hypothetical of [(C5)v-(C6)w]2, each instance of v and w may be an integer from 0 to 2 as described above. Thus, the first instance of v and the second instance of v may each be 1, or the first instance of v may be 1 and the second instance of v may be 2.
Thus, for example, when considering the formula of Formula III C1-(C2)r-(C3)t-(C4)u-[(C5)v-(C6)w]x-(C7)y-(C8)z-(C9)-(C10)-(C11)-[(C12)-(C13)]a wherein x is 3, one can also envision the formula of Formula III to be written as:
C1-(C2)r-(C3)t-(C4)u-(C5)v-(C6)w-(C5)v-(C6)w-(C5)v-(C6)w-(C7)y-(C8)z-(C9)—(C10)-(C11)-[(C12)-(C13)]a
wherein each v and w are selected, independently, from 0, 1 or 2, and each C5 and C6 are selected, independently, from an appropriate amino acid as outlined above. This meaning, unless explicitly indicated otherwise, expands to all further formulas disclosed herein and below.
In some embodiments, the sequence of SEQ ID NO. 9 can be derived from Formula III as follows: r is 1, t is 2, u is 2, v is 2, w is 2, x is 2, y is 2, z is 1, and a is 1; C1 is methionine, C2 is K, the string of two (2) C3 residues is as follows: L-S, the string of two (2) C4 residues is as follows: S-L, the string of eight (8) residues given by [(C5)2-(C6)2]2 is as follows: L-L-A-L-L-L-A-L (SEQ ID NO: 89), the string of two (2) C7 residues is as follows: A-S, C8 is L, C9 is A, C10 is L, C11 is A, C12 is present and is A, and C13 is present and is P.
In some embodiments, the pre-protein signal peptide comprises an amino acid sequence represented by:
D1-(D2)q-(D3)r-(D4)t-(D5)u-[(D6)v-(D7)x-(D8)w-(D9)y]z-(D10)-(D11)-(D12)-[(D13)-(D14)]a (Formula IV)
wherein D2-D14 have the properties described in Table 6 below:
5-10.8
−4-34
1-1.3
5-10.8
and q is an integer selected from 1, 2, or 3 (inclusive);
In some embodiments, D1 is methionine. In some embodiments, each D2 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each D3 is, independently, an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 89 g/mol to about 205 g/mol, a hydropathy index of about −4 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each D4, D9 and D11 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each D4 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each D9 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, D11 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each D5 is, independently, an amino acid having an isoelectric point of about 3.2 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.75 to about 1.3. In some embodiments, each D6 is, independently, an amino acid having an isoelectric point from about 5 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each D7 is, independently, an amino acid having an isoelectric point of about 5.4 to about 6.1, a molecular weight of about 117 g/mol to about 205 g/mol, a hydropathy index of about 2.5 to about 34, and a helicity of about 1 to about 1.3. In some embodiments, each D8, D10, D12, and D13 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 182 g/mol, a hydropathy index of about −5.1 to about 32, and a helicity of about 0.75 to about 1.3. In some embodiments, each D8 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 182 g/mol, a hydropathy index of about −5.1 to about 32, and a helicity of about 0.75 to about 1.3. In some embodiments, D10 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 182 g/mol, a hydropathy index of about −5.1 to about 32, and a helicity of about 0.75 to about 1.3. In some embodiments, D12 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 182 g/mol, a hydropathy index of about −5.1 to about 32, and a helicity of about 0.75 to about 1.3. In some embodiments, D13 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 182 g/mol, a hydropathy index of about −5.1 to about 32, and a helicity of about 0.75 to about 1.3. In some embodiments, D14 is an amino acid with an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 182 g/mol, a hydropathy index of about −5.1 to about 32, and a helicity of about 0.5 to about 1.3.
In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, r is 0. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, y is 0. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, z may be an integer selected from 3-9, 4-8, 6-10, 8-10, 2-5, or 3-6 (all inclusive). In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, z is 4. In some embodiments, z is 5. In some embodiments, z is 6. In some embodiments, z is 7. In some embodiments, z is 8. In some embodiments, z is 9. In some embodiments, z is 10. In some embodiments a is 0 and the residues given by [(D13)-(D14)]a are absent. In some embodiments, a is 1 and the residues given by [(D13)-(D14)]a are present. It is to be understood that the values of r, t, u, v, w, x, y, and z are each independently selected, and the value of any variable r, t, u, v, w, x, y, or z is independent of the values selected for the other variables. In some embodiments, each D2 is, independently, an amino acid selected from the group consisting of K and R. In some embodiments, each D3 is, independently, an amino acid selected from the group consisting of F, L, I, W, V, M, Y, P, C, A, Q, and S. In some embodiments, each D4, D9 and D11 is each, independently, an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, each D4, D9 and D11 is each, independently, an amino acid selected from the group consisting of L and I. In some embodiments, each D4 is, independently, an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, each D4 is, independently, an amino acid selected from the group consisting of L or I. In some embodiments, each D9 is, independently, an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, each D9 is, independently, an amino acid selected from the group consisting of L and I. In some embodiments, D9 is an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, D9 is an amino acid selected from the group consisting of L and I. In some embodiments, D11 is an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, D11 is an amino acid selected from the group consisting of L and I. In some embodiments, each D5 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, G, K, E, H, A, C, Y, V, W, I, F, and L. In some embodiments, each D8, D10, D12, and D13 is each, independently, an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, each D8, D10, D12, and D13 is each, independently, an amino acid selected from the group consisting of A and S. In some embodiments, each D8 is, independently, an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, each D8 is, independently, an amino acid selected from the group consisting of A and S. In some embodiments, D10 is an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, D10 is an amino acid selected from the group consisting of A and S. In some embodiments, D12 is an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, D12 is an amino acid selected from the group consisting of A and S. In some embodiments, D13 is an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, D13 is an amino acid selected from the group consisting of A and S. In some embodiments, each D7 is, independently, an amino acid selected from the group consisting of V, W, I, L, F, and T. In some embodiments, each D6 is, independently, an amino acid selected from the group consisting of L, I, A, T, S, G, N, R K, Y, Q, C, H, W, and M. In some embodiments, each D6 is, independently, an amino acid selected from the group consisting of L and I. In some embodiments, D14 is an amino acid selected from the group consisting of P, Y, M, V, A, T, Q, S, N, G, I, E, D, L, F, R, K, and H. In embodiments where any one of r, t, u, v, w, x, y, and z are an integer greater than 1, each amino acid in the group described by the r, t, u, v, w, x, y, and z are independently chosen from the disclosed group of amino acids and therefore may be the same or different, as described for herein.
As outlined pertaining to Formula III, the portion of Formula IV given by -[(D6)v-(D7)x-(D8)w-(D9)y]z is not to be interpreted as “z” repeats of [(D6)v-(D7)x-(D8)w-(D9)y], but rather, when expanded “z” times, each v, x, w, and y may be independently selected from an integer as provided for above, and each D6, D7, D8, and D9 may be independently selected from an appropriate amino acid as provided for above.
Thus, for example, when considering the formula of Formula IV D1-(D2)q-(D3)r-(D4)t-(D5)u-[(D6)v-(D7)x-(D8)w(D9)y]z-(D10)-(D11)-(D12)-[(D13)-(D14)]a wherein z is 3, one can also envision the formula of Formula IV to be written as: D1-(D2)q-(D3)r-(D4)t-(D5)u-(D6)v-(D7)x-(D8)w-(D9)y-(D6)v-(D7)x-(D8)w-(D9)y-(D6)v-(D7)x-(D8)w-(D9)y-(D10)-(D11)-(D12)-[(D13)-(D14)]a wherein each v, x, w, and y are selected, independently, from 0, 1 or 2, and each D6, D7, D8, and D9 are selected, independently, from an appropriate amino acid as outlined above.
In some embodiments, the sequence of SEQ ID NO. 12 can be derived from Formula IV as follows: q is 1, r is 1, t is 1, u is 2, for every instance of z v is 0, for every instance of z x is 0, w is 1, y is 1, z is 6, and a is 1; D1 is methionine; D2 is K; D3 is F; D4 is L; the string of two (2) D5 residues is as follows: S-L; for every instance of z D6 is absent; for every instance of z D7 is absent; the string of twelve (12) residues given by [(D8)1-(D9)1]6 is as follows: L-L-A-L-V-A-A-L-A-L-A-L (SEQ ID NO: 90); D10 is A; D11 is L; D12 is A; D13 is present and is A; and D14 is present and is P.
In some embodiments, the pre-protein signal peptide comprises an amino acid sequence represented by:
E1-[(E2)i(E3)j-(E4)q]r-(E5)t-(E6)u-(E7)v-[(E8)w-(E9)x]y-(E10)z-(E11)-(E12)-(E13)-[(E14)-(E15)]a Formula V
wherein E2-E15 have the properties described in Table 7 below:
5-10.8
5-10.8
5-9.75
−4-33.5
5-10.8
5-9.75
−4-33.5
−4-15.5
wherein each i, j, q, w, x, and a are independently 0 or 1;
In some embodiments, E1 is methionine. In some embodiments, each E2 is, independently, an amino acid having an isoelectric point of about 3.2 to about 10.8, a molecular weight of about 105 g/mol to about 175 g/mol, a hydropathy index of about −4 to about 1, and a helicity of about 0.85 to about 1. In some embodiments, each E3 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75.1 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, each E4 is, independently, an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 105 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, each E5 and E8 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, each E5 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, each E8 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, each E6 is, independently, an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 89 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, each E7 is, independently, an amino acid having an isoelectric point of about 5 to about 9.75, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −4 to about 33.5, and a helicity of about 0.79 to about 1.3. In some embodiments, each E9, E13, and E14 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, each E9 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, E13 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, E14 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. In some embodiments, each E10 and E12 is, independently, an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each E10 is, independently, an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, E12 is an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, E11 is an amino acid having an isoelectric point of about 5 to about 9.75, a molecular weight of about 89 g/mol to about 205 g/mol, a hydropathy index of about −4 to about 33.5, and a helicity of about 0.79 to about 1.3. In some embodiments, E15 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol, a hydropathy index of about −4 to about 15.5, and a helicity of about 0.57 to about 1.2.
In some embodiments, i is 0. In some embodiments, i is 1. In some embodiments, j is 0. In some embodiments, j is 1. In some embodiments, q is 0. In some embodiments, q is 1. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, z is 0. In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, y may be an integer selected from 3-9, 4-8, 6-10, 8-10, 2-5, or 3-6 (all inclusive). In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7. In some embodiments, y is 8. In some embodiments, y is 9. In some embodiments, y is 10. In some embodiments a is 0 and the residues given by [(E14)-(E15)]a are absent. In some embodiments, a is 1 and the residues given by [(E14)-(E15)]a are present. It is to be understood that the values of i, j, q, w, x, r, t, u, v, z, and y are each independently selected, and the value of any variable i, j, q, w, x, r, t, u, v, z, or y is independent of the values selected for the other variables. In some embodiments, each E2 is, independently, an amino acid selected from the group consisting of K, R, S, Q, and E. In some embodiments, each E3 is, independently, an amino acid selected from the group consisting of F, L, I, W, V, Y, P, A, T, Q, N, S, G, D, R, K, and H. In some embodiments, each E3 is, independently, an amino acid selected from the group consisting of F, L, I, W, V, and Y. In some embodiments, each E4 is, independently, an amino acid selected from the group consisting of K, R, H, S, C, P, Y, M, V, W, I, L, and F. In some embodiments, each E4 may independently be K, R, H, and S. In some embodiments, each E5 and E8 is each, independently, an amino acid selected from the group consisting of L, I, F, V, C, A, Y, T, Q, N, S, K, H, W, G, D, M, P, E, and R. In some embodiments, each E5 and E8 is each, independently, an amino acid selected from the group consisting of L, I, F, V, and C. In some embodiments, each E5 is, independently, an amino acid selected from the group consisting of L, I, F, V, C, A, Y, T, Q, N, S, K, H, W, G, D, M, P, E, and R. In some embodiments, each E5 is, independently, an amino acid selected from the group consisting of L, I, F, V, and C. In some embodiments, each E8 is, independently, an amino acid selected from the group consisting of L, I, F, V, C, A, Y, T, Q, N, S, K, H, W, G, D, M, P, E, and R. In some embodiments, each E8 is, independently, an amino acid selected from the group consisting of L, I, F, V, and C. In some embodiments, each E6 is, independently, an amino acid selected from the group consisting of T, Q, S, A, C, R, K, H, P, V, W, I, F, and L. In some embodiments, each E7 is, independently, an amino acid selected from the group consisting of S, G, K, A, C, Y, V, and W. In some embodiments, each E9, E13, and E14 is each, independently, an amino acid selected from the group consisting of A, T, G, S, V, I, L, Y, W, F, C, Q, N, P, E, M, R, K, D, and H. In some embodiments, each E9, E13, and E14 is each, independently, an amino acid selected from the group consisting of A, T, G, S, V, I, and L. In some embodiments, each E9 is, independently, an amino acid selected from the group consisting of A, T, G, S, V, I, L, Y, W, F, C, Q, N, P, E, M, R, K, D, and H. In some embodiments, each E9 is, independently, an amino acid selected from the group consisting of A, T, G, S, V, I, and L. In some embodiments, each E10 and E12 is, independently, an amino acid selected from the group consisting of L, F, I, V, C, Y, T, Q, N, S, K, H, M, G, A, W, D, P, E, and R. In some embodiments, each E10 and E12 is, independently, an amino acid selected from the group consisting of L, F, I, V, and C. In some embodiments, each E10 is, independently, an amino acid selected from the group consisting of L, F, I, V, C, Y, T, Q, N, S, K, H, M, G, A, W, D, P, E, and R. In some embodiments, each E10 is, independently, an amino acid selected from the group consisting of L, F, I, V, and C. In some embodiments, E12 is an amino acid selected from the group consisting of L, F, I, V, C, Y, T, Q, N, S, K, H, M, G, A, W, D, P, E, and R. In some embodiments, E12 is an amino acid selected from the group consisting of L, F, I, V, and C. In some embodiments, E13 is an amino acid selected from the group consisting of A, T, G, S, V, I, L, Y, W, F, C, Q, N, P, E, M, R, K, D, and H. In some embodiments, E13 is an amino acid selected from the group consisting of A, T, G, S, V, I, and L. In some embodiments, E14 is an amino acid selected from the group consisting of A, T, G, S, V, I, L, Y, W, F, C, Q, N, P, E, M, R, K, D, and H. In some embodiments, E14 is an amino acid selected from the group consisting of A, T, G, S, V, I, and L. In some embodiments, each E11 is, independently, an amino acid selected from the group consisting of V, W, I, C, L, A, T, S, and K. In some embodiments, each E15 is, independently, an amino acid selected from the group consisting of S, N, R, T, G, K, E, D, P, and Y. In embodiments where any one of r, t, u, v, z, and y are an integer greater than 1, each amino acid in the group described by the r, t, u, v, z, and y are independently chosen from the disclosed group of amino acids and therefore may be the same or different, as described for herein.
As outlined pertaining to Formula III, the portion of Formula V given by [(E8)w-(E9)x]y is not to be interpreted as “y” repeats of [(E8)w-(E9)x], but rather, when expanded “y” times, each w and x may be independently selected from an integer as provided for above, and each E8 and E9 may be independently selected from an appropriate amino acid as provided for above. The same is to be understood for the portion of Formula V given by [(E2)i-(E3)j-(E4)q]r.
Thus, for example, when considering the formula of Formula V, E1-[(E2)i-(E3)j-(E4)q]r-(E5)t-(E6)u-(E7)v-[(E8)w(E9)x]y-(E10)z-(E11)-(E12)-(E13)-[(E14)-(E15)]a, wherein r is 2 and y is 2, one can also envision the formula of Formula V to be written as: E1-(E2)i-(E3)j-(E4)q-(E2)i-(E3)j-(E4)q-(E5)t-(E6)u-(E7)v-(E8)w-(E9)x-(E8)w(E9)x-(E10)z-(E11)-(E12)-(E13)-[(E14)-(E15)]a wherein each i, j, q, w and x are selected, independently, from 0 or 1, and each E2, E3, E4, E5, and E9 are selected, independently, from an appropriate amino acid as outlined above.
In some embodiments, the sequence of SEQ ID NO. 14 can be derived from Formula V as follows: i is 1, j is 1, q is 1, r is 1, t is 1, u is 2, v is 0, w is 1, x is 1, y is 5, z is 0, and a is 1; E1 is methionine; E2 is K; E3 is F; E4 is K; E5 is L; the string of two (2) E6 residues is as follows: T-L; E7 is absent; the string of ten (10) residues given by [(E8)1-(E9)1]5 is as follows: L-A-A-L-L-A-L-A-A-L (SEQ ID NO: 91); E10 is absent; E11 is V; E12 is L; E13 is A; E14 is present and is A; and E15 is present and is S.
In some embodiments, the sequence of SEQ ID NO. 15 can be derived from Formula V as follows: i is 1, j is 1, q is 1, r is 1, t is 1, u is 2, v is 0, w is 1, x is 1, y is 4, z is 0, and a is 1; E1 is methionine; E2 is K; E3 is L; E4 is S; E5 is S; the string of two (2) E6 residues is as follows: I-L; E7 is absent; the string of eight (8) residues given by [(E8)1-(E9)1]4 is as follows: L-L-L-A-L-L-A-L (SEQ ID NO: 92); E10 is absent; En is V; E12 is L; E13 is A; E14 is present and is A; and E15 is present and is S.
In some embodiments, the sequence of SEQ ID NO. 16 can be derived from Formula V as follows: i is 1, j is 1, q is 1, r is 2, t is 1, u is 2, v is 0, w is 1, x is 1, y is 3, z is 0, and a is 1; E1 is methionine; the string of six (6) residues given by [(E2)1-(E3)1-(E4)1]2 is as follows: K-L-L-S-L-L (SEQ ID NO: 106); E5 is A; the string of two (2) E6 residues is as follows: L-L; E7 is absent; the string of six (6) residues given by [(E8)1-(E9)1]3 is as follows: L-L-L-A-S-L (SEQ ID NO: 93); E10 is absent; E11 is V; E12 is L; E13 is A; E14 is present and is A; and E15 is present and is S.
In some embodiments, the pre-protein signal peptide comprises an amino acid sequence represented by:
F1-(F2)v-(F3)w-[(F4)x-(F5)y]z-(F6)-(F7)-(F8)-[(F9)-(F10)]a (Formula IX)
wherein F1-F10 have the properties described in Table 8 below:
−4-31
wherein v and w are independently integers selected from 0, 1, 2, or 3 (inclusive); and
In some embodiments, F1 is an amino acid having an isoelectric point of about 5.4 to about 11, a molecular weight of about 89 g/mol to about 175 g/mol; a hydropathy index of about −4 to about 31, and a helicity of about 0.9 to about 1.3. In some embodiments, each F2 is, independently, an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each F3 and F7 is each, independently, an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each F3 is, independently, an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, F7 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each F4 is, independently, an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each F5, F6, F8, and F9 is each, independently, an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, each F5 is, independently, an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, F6 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, F8 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, F9 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3. In some embodiments, F10 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity of about 0.5 to about 1.3.
In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, y is 0. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, z may be an integer selected from 3-8, 4-8, 6-8, 2-5, or 3-6 (all inclusive). In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, z is 4. In some embodiments, z is 5. In some embodiments, z is 6. In some embodiments, z is 7. In some embodiments, z is 8. In some embodiments a is 0 and the residues given by [(F9)-(F10)]a are absent. In some embodiments, a is 1 and the residues given by [(F9)-(F10)]a are present. It is to be understood that the values of v, w, x, y, and z are each independently selected, and the value of any variable v, w, x, y, or z is independent of the values selected for the other variables. In some embodiments, F1 is an amino acid selected from the group consisting of M, F, L, A, S, or R. In some embodiments, each F2 is, independently, an amino acid selected from the group consisting of K, R, H, S, G, N, Q, E, T, A, C, P, Y, V, W, I, L, and F. In some embodiments, each F2 is, independently, an amino acid selected from the group consisting of K, R, H, S, G, N, Q, E, T, and A. In some embodiments, each F3 and F7 is, independently, an amino acid selected from the group consisting of S, Q, R, T, K, H, I, F, L, P, N, G, E, D, A, Y, M, V, W, and C. In some embodiments, each F3 and F7 is, independently an amino acid selected from the group consisting of S, Q, R, T, K, H, I, F, and L. In some embodiments, each F4 is, independently, an amino acid selected from the group consisting of L, I, V, M, A, F, W, Y, P, C, T, Q, N, S, G, E, R, K, and H. In some embodiments, each F4 is, independently, an amino acid selected from the group consisting of L, I, V, M, and A. In some embodiments, each F5, F6, F8, and F9 is each, independently, an amino acid selected from the group consisting of A, C, G, S, V, L, T, F, Q, N, P, Y, E, K, H, W, I, M, and R. In some embodiments, each F5, F6, F8, and F9 is each, independently, an amino acid selected from the group consisting of A, C, G, S, V, and L. In some embodiments, F10 is an amino acid selected from the group consisting of P, C, Y, M, V, A, T, Q, S, N, W, G, I, E, L, F, R, K, and H. In embodiments where any one of v, w, x, y, and z are an integer greater than 1, each amino acid in the group described by the v, w, x, y, and z are independently chosen from the disclosed group of amino acids and therefore may be the same or different, as described for herein.
As outlined pertaining to Formula III, the portion of Formula IX given by [(F4)x-(F5)y]z is not to be interpreted as “z” repeats of [(F4)x-(F5)y], but rather, when expanded “z” times, each x and y may be independently selected from an integer as provided for above, and each F4 and F5 may be independently selected from an appropriate amino acid as provided for above.
Thus, for example, when considering the formula of Formula IX F1-(F2)v-(F3)w-[(F4)x-(F5)y]z-(F6)—(F7)-(F8)-[(F9)—(F10)]a wherein z is 3, one can also envision the formula of Formula IX to be written as: F1-(F2)v-(F3)w-(F4)x-(F5)y-(F4)x-(F5)y-(F4)x-(F5)y-(F6)-(F7)-(F5)-[(F9)-(F10)]a wherein each x and y are selected, independently, from 0, 1, 2, 3, or 4, and each F4 and F5 are selected, independently, from an appropriate amino acid as outlined above.
In some embodiments, the sequence of SEQ ID NO. 31 can be derived from Formula IX as follows: v is 3, w is 0, x is 1, y is 1, z is 6, and a is 1; F1 is methionine; the string of three (3) F2 residues is as follows: K-S-S; F3 is absent; the string of twelve (12) residues given by [(F4)1-(F5)1]6 is as follows: L-L-L-L-A-L-L-A-L-A-A-L (SEQ ID NO: 94); F6 is A; F7 is S; F8 is A; F9 is present and is A; and F10 is present and is P.
In some embodiments, the sequence of SEQ ID NO. 32 can be derived from Formula IX as follows: v is 2, w is 0, x is 1, y is 1, z is 6, and a is 1; F1 is methionine; the string of two (2) F2 residues is as follows: K-S; F3 is absent; the string of twelve (12) residues given by [(F4)1-(F5)1]6 is as follows: S-L-L-L-L-L-L-A-L-A-S-L (SEQ ID NO: 95); F6 is A; F7 is L; F8 is A; F9 is present and is A; and F10 is present and is P.
In some embodiments, the sequence of SEQ ID NO. 33 can be derived from Formula IX as follows: v is 3, w is 0, x is 1, y is 1, z is 7, and a is 1; F1 is methionine; the string of three (3) F2 residues is as follows: K-S-S; F3 is absent; the string of fourteen (14) residues given by [(F4)1-(F5)1]7 is as follows: S-L-L-L-L-A-L-L-A-L-L-A-A-L (SEQ ID NO: 96); F6 is A; F7 is S; F8 is A; F9 is present and is A; and F10 is present and is P.
In some embodiments, the pre-protein signal peptide comprises an amino acid sequence represented by:
L1-(L2)x-[(L3)a-(L4)a]y-[(L5)a-(L6)a-(L7)a]z-(L8)a-(L9)a-(L10)a-(L11)a-(L12)a (Formula XIII)
wherein L2-L12 have the properties described in Table 9 below:
wherein:
In some embodiments, L1 is methionine. In some embodiments, each L2 is, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, each L3 and L6 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, each L3 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, each L6 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, each L4, L7 and L9 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, each L4 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, each L7 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, L9 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, each L5, L8, L10 and L11 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, each L5 is each, independently, an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, L8 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, L10 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, L11 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3. In some embodiments, L12 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g/mol to about 205 g/mol; a hydropathy index of about −5.1 to about 34, and a helicity or about 0.5 to about 1.3.
In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, z is 5. In some embodiments, z is 6. In some embodiments, z is 7. In some embodiments, z is 8. In some embodiments, z is 9. In some embodiments, z is 10. In some embodiments, a is 0. In some embodiments, a is 1. It is to be understood that the values of any variable x, y, z, and a are each independently selected, and the value of any variable x, y, z, or a is independent of the value selected for the other variables. In some embodiments, L1 is methionine. In some embodiments, each L2 is, independently, an amino acid selected from the group consisting of R, K, H, S, G, N, Q, D, T, A, C, P, Y, M, V, W, I, F, and L. In some embodiments, each L2 is, independently, an amino acid selected from the group consisting of R, K, and H. In some embodiments, L3 is absent. In some embodiments, L3 is present. In some embodiments, each L3 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, P, G, E, H, D, A, C, Y, M, V, W, I, F, and L. In some embodiments, each L3 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, and P. In some embodiments, L4 is absent. In some embodiments, L4 is present. In some embodiments, each L4 is, independently, an amino acid selected from the group consisting of L, F, I, W, V, T, M, Y, P, C, A, Q, N, S, G, E, D, R, K, and H. In some embodiments, each L4 is, independently, an amino acid selected from the group consisting of L, F, I, W, V, and T. In some embodiments, L5 is absent. In some embodiments, L5 is present. In some embodiments, each L5 is, independently, an amino acid selected from the group consisting of A, T, G, S, C, P, I, L, F, R, V, Q, Y, K, N, E, D, H, M, and W. In some embodiments, each L5 is, independently, an amino acid selected from the group consisting of A, T, G, and S. In some embodiments, L6 is absent. In some embodiments, L6 is present. In some embodiments, each L6 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, P, G, E, H, D, A, C, Y, M, V, W, I, F, and L. In some embodiments, each L6 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, K, and P. In some embodiments, L7 is absent. In some embodiments, L7 is present. In some embodiments, each L7 is, independently, an amino acid selected from the group consisting of L, F, I, W, V, T, M, Y, P, C, A, Q, N, S, G, E, D, R, K, and H. In some embodiments, each L7 is, independently, an amino acid selected from the group consisting of L, F, I, W, V, and T. In some embodiments, L8 is absent. In some embodiments, L8 is present. In some embodiments, L8 is an amino acid selected from the group consisting of A, T, G, S, C, P, I, L, F, R, V, Q, Y, K, N, E, D, H, M, and W. In some embodiments L8 is an amino acid selected from the group consisting of A, T, G, and S. In some embodiments, L9 is absent. In some embodiments, L9 is present. In some embodiments, L9 is an amino acid selected from the group consisting of L, F, I, W, V, T, M, Y, P, C, A, Q, N, S, G, E, D, R, K, and H. In some embodiments, L9 is an amino acid selected from the group consisting of L, F, I, W, V, and T. In some embodiments, L10 is absent. In some embodiments, L10 is present. In some embodiments, L10 is an amino acid selected from the group consisting of A, T, G, S, C, P, I, L, F, R, V, Q, Y, K, N, E, D, H, M, and W. In some embodiments, L10 is an amino acid selected from the group consisting of A, T, G, and S. In some embodiments, L11 is absent. In some embodiments, L1 is present. In some embodiments, Ln is an amino acid selected from the group consisting of A, T, G, S, C, P, I, L, F, R, V, Q, Y, K, N, E, D, H, M, and W. In some embodiments Ln is an amino acid selected from the group consisting of A, T, G, and S. In some embodiments, L12 is absent. In some embodiments, L12 is present. In some embodiments, L12 is an amino acid selected from the group consisting of P, T, S, D, C, Y, M, V, A, Q, N, W, G, I, E, L, F, R, K, and H. In some embodiments L12 is an amino acid selected from the group consisting of P, T, S, and D. In embodiments where any one of x, y, and z are an integer greater than 1, each amino acid in the group described by the x, y, and z are independently chosen from the disclosed group of amino acids and therefore may be the same or different, as described for herein.
As outlined pertaining to Formula III, the portion of Formula XIII given by [(L5)a-(L6)a-(L7)a]z is not to be interpreted as “z” repeats of [(L5)a-(L6)a-(L7)a], but rather, when expanded “z” times, each a may be independently selected from an integer as provided for above, and each L5, L6, and L7 may be independently selected from an appropriate amino acid as provided for above.
Thus, for example, when considering the formula of Formula XIII L1-(L2)x-[(L3)a-(L4)a]y-[(L5)a-(L6)a-(L7)a]z-(L8)a-(L9)a-(L10)a-(L11)a-(L12)a wherein z is 5, one can also envision the formula of Formula XIII to be written as: L1-(L2)x-[(L3)a-(L4)a]y-(L5)a-(L6)a-(L7)a-(L5)a-(L6)a-(L7)a-(L5)a-(L6)a-(L7)a-(L5)a-(L6)a-(L7)a-(L5)a (L6)a-(L7)a-(L8)a-(L9)a-(L10)a-(L11)a-(L12)a wherein each a is, independently, 0 or 1 and each L5, L6, and L7 are selected, independently, from an appropriate amino acid as outline above.
In some embodiments, the sequence of SEQ ID NO. 70 can be derived from Formula XIII as follows: x is 1, y is 2, and z is 6; L1 is methionine; L2 is R; all four instances of “a” within [(L3)a-(L4)a]2 are 1 and the string of four (4) residues given by [(L3)1-(L4)1]2 is as follows: S-L-S-L; for every (L5)a, “a” is 1; for every (L6)a, “a” is 0; for every (L7)a, “a” is 1; the string of twelve (12) residues given by [(L5)1-(L7)1]6 is as follows: A-L-L-L-L-L-A-L-L-A-S-L (SEQ ID NO: 97); L6 is absent; L8 is present and is A; L9 is present and is L; L10 is present and is A; L11 is present and is A; L12 is present and is P.
In some embodiments, the sequence of SEQ ID NO. 71 can be derived from Formula XIII as follows: x is 1, y is 2, and z is 6; L1 is methionine; L2 is R; all four instances of “a” within [(L3)a-(L4)a]2 are 1 and the string of four (4) residues given by [(L3)1-(L4)1]2 is as follows: L-S-L-S; for every (L5)a, “a” is 1; for every (L6)a, “a” is 0; for every (L7)a, “a” is 1; the string of twelve (12) residues given by [(L5)1-(L7)1]6 is as follows: L-L-L-L-L-L-A-L-L-A-S-L (SEQ ID NO: 98); L6 is absent; L8 is present and is A; L9 is present and is L; L10 is present and is A; L11 is present and is A; L12 is present and is P.
In some embodiments, the sequence of SEQ ID NO. 72 can be derived from Formula XIII as follows: x is 1, y is 2, and z is 6; L1 is methionine; L2 is R; all four instances of “a” within [(L3)a-(L4)a]2 are 1 and the string of four (4) residues given by [(L3)1-(L4)1]2 is as follows: L-S-S-L; for every (L5)a, “a” is 1; for every (L6)a, “a” is 0; for every (L7)a, “a” is 1; the string of twelve (12) residues given by [(L5)1-(L7)1]6 is as follows: L-L-G-L-L-L-A-L-A-A-S-L (SEQ ID NO: 99); L6 is absent; L8 is present and is A; L9 is present and is L; L10 is present and is A; L11 is present and is A; L12 is present and is P.
In some embodiments, the sequence of SEQ ID NO. 73 can be derived from Formula XIII as follows: x is 1, y is 1, and z is 7; L1 is methionine; L2 is R; both instances of “a” within [(L3)a-(L4)a]2 are 1 and the string of two (2) residues given by [(L3)1-(L4)1]1 is as follows: L-S; for every (L5)a, “a” is 1; for every (L6)a, “a” is 0; for every (L7)a, “a” is 1; the string of fourteen (14) residues given by [(L5)1-(L7)1]7 is as follows: L-L-L-A-L-L-A-L-L-A-L-A-S-L (SEQ ID NO: 100); L6 is absent; L8 is present and is A; L9 is present and is L; L10 is present and is A; L11 is present and is A; L12 is present and is P.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence represented by:
G1-G2-G3-G4-G5-G6-G7-G8-G9-G10-G11-G12-G13-G14-G15-G16-G17-G18-G19-G20-G21-G22-G23-G24-G25 (Formula VI)
wherein Table 10 below describes the various substitutions that may be made, with preferable amino acids underlined.
−4-34
−4-14
−4-10
−1-14
−4-34
24-31
−4-23
−1-25
V, P, Y, I, A, E, K, F, T,
−4-31
−5-31
In some embodiments, G1 is an amino acid selected from the group consisting of I, L, F, V, A, N, S, D, R, and K. In some embodiments, G2 is an amino acid selected from the group consisting of P, S, N, G, and E. In some embodiments, G3 is an amino acid selected from the group consisting of L, F, I, V, Y, A, S, R, and H. In some embodiments, G4 is an amino acid selected from the group consisting of V, M, P, Y, A, T, S, N, K, and H. In some embodiments, G5 is an amino acid selected from the group consisting of A, G, R, Y, K, D, M, V, W, I, and L. In some embodiments, G6 is an amino acid selected from the group consisting of N, R, and K. In some embodiments, G7 is an amino acid selected from the group consisting of V, P, A, T, Q, G, E, D, R, and K. In some embodiments, G8 is an amino acid selected from the group consisting of P, Y, T, Q, S, N, W, F, R, K, and H. In some embodiments, G9 is an amino acid selected from the group consisting of F, L, A, Q, N, S, E, G, D, and H. In some embodiments, G10 is an amino acid selected from the group consisting of H, S, N, D, Q, E, T, Y, M, V, I, and L. In some embodiments, G11 is an amino acid selected from the group consisting of S, R, T, G, K, E, D, and P. In some embodiments, G12 is an amino acid selected from the group consisting of D, E, Q, N, A, and V. In some embodiments, G13 is an amino acid selected from the group consisting of N, S, E, D, T, H, K, A, and P. In some embodiments, G14 is an amino acid selected from the group consisting of G, S, N, H, E, C, Y, L, and F. In some embodiments, G15 is an amino acid selected from the group consisting of S, T, and H. In some embodiments, G16 is an amino acid selected from the group consisting of E, D, Q, N, S, T, K, and A. In some embodiments, G17 is an amino acid selected from the group consisting of W, N, D, and R. In some embodiments, G18 is an amino acid selected from the group consisting of L and F. In some embodiments, G19 is an amino acid selected from the group consisting of Y, V, A, Q, N, S, E, D, L, R, K, and H. In some embodiments, G20 is an amino acid selected from the group consisting of K, R, S, and I. In some embodiments, G21 is R. In some embodiments, G22 is an amino acid selected from the group consisting of D, E, N, S, T, G, A, Y, and L. In some embodiments, G23 is an amino acid selected from the group consisting of V, P, Y, I, A, E, K, F, T, S, G, D, M, and N. In some embodiments, G23 is an amino acid selected from the group consisting of V, P, Y, I, A, E, and K. In some embodiments, G24 is an amino acid selected from the group consisting of V, P, Y, I, A, E, K, F, T, S, G, D, M, and N. In some embodiments, G24 is an amino acid selected from the group consisting of V, P, Y, I, A, E, and K. In some embodiments, G25 is an amino acid selected from the group consisting of Y, P, A, T, Q, S, E, F, and H.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO. 86 (IPLVANVSFNSDNGSQWLYKRDVVY).
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence represented by:
(H1)m-(H2)m-(H3)m-(H4)m-(H5)m-(H6)m-(H7)m-(H8)m-(H9)m-(H10)m-(H11)m- (H12)m-(H13)m-(H14)m-(H15)m-(H16)m-(H17)m-(H18)m-(H19)m-(H20)m-(H21)m-(H22)m-(H23)m-(H24)m-(H25)m-(H26)m-(H27)m-(H28)m-(H29)m-(H30)m-(H31)m-(H32)m-(H33)m-(H34)m-(H35)m-(H36)m-H37-H38-H39-H40 (Formula VII)
wherein each m is, independently, 0, 1, or 2. Table 11 below describes the various amino acids that may be used at each position, with preferable amino acids underlined.
E, D, S, L, G, Q, A
P, S. R, T, N, G, D, K, A
T, Q, A, E, F, S
T, G, V, W, A
1-1.3
E, S, D, L, G
1-1.3
S, K, T, A, Y, M, F
A, T, G, R, Y, L, F, E
V, I, F, M, L, A, T
1-1.3
In some embodiments, amino acid positions H1-H36 may be omitted or repeated up to 1 extra time (i.e., be included 0 to 2 times), each repeat being independently selected from the indicated amino acids. Further, it is to be understood that the omission or repetition of any amino acid positions H1-H36 is independent of the omission or repetition of any amino acid at an alternate position. In some embodiments, the minimum length of a sequence generated with Formula VII is fourteen (14) amino acids.
In some embodiments, each H1 is, independently, absent. In some embodiments, each H1 is, independently, an amino acid selected from the group consisting of E, D, S, L, G, Q, and A. In some embodiments, each H1 is, independently, an amino acid selected from the group consisting of E, D, and S. In some embodiments, each H2 is, independently, absent. In some embodiments, each H2 is, independently, an amino acid selected from the group consisting of P, S, R, T, N, G, D, K, and A. In some embodiments, each H2 is, independently, an amino acid selected from the group consisting of P, S, and R. In some embodiments, each H3 is, independently, absent. In some embodiments, each H3 is, independently, an amino acid selected from the group consisting of W and Y. In some embodiments, each H4 is, independently, absent. In some embodiments, each H4 is, independently, an amino acid selected from the group consisting of S, N, A, P, and V. In some embodiments, each H5 is, independently, absent. In some embodiments, each H5 is, independently, an amino acid selected from the group consisting of T, Q, A, E, F, and S. In some embodiments, each H5 is, independently, T. In some embodiments, each H6 is, independently, absent. In some embodiments, each H6 is, independently, an amino acid selected from the group consisting of L, F, and I. In some embodiments, each H7 is, independently, absent. In some embodiments, each H7 is, independently, an amino acid selected from the group consisting of F, V, M, T, S, and K. In some embodiments, each H8 is, independently, absent. In some embodiments, each H8 is, independently, an amino acid selected from the group consisting of V, P, I, A, S, and K. In some embodiments, each H9 is, independently, absent. In some embodiments, each H9 is, independently, an amino acid selected from the group consisting of T, G, V, W, and A. In some embodiments, each H9 is, independently, an amino acid selected from the group consisting of T, G, and V. In some embodiments, each H10 is, independently, absent. In some embodiments, each H10 is, independently, an amino acid selected from the group consisting of R, H, S, G, N, E, T, and V. In some embodiments, each H11 is, independently, absent. In some embodiments, each H11 is, independently, an amino acid selected from the group consisting of S, G, D, A, and M. In some embodiments, each H12 is, independently, absent. In some embodiments, each H12 is, independently, an amino acid selected from the group consisting of T, S, E, G, D, K, and H. In some embodiments, each H13 is, independently, absent. In some embodiments, each H13 is, independently, an amino acid selected from the group consisting of L, M, Y, N, S, D, and K. In some embodiments, each H14 is, independently, absent. In some embodiments, each H14 is, independently, an amino acid selected from the group consisting of D, Q, N, S, K, and C. In some embodiments, each His is, independently, absent. In some embodiments, each His is, independently, an amino acid selected from the group consisting of E, S, D, L, and G. In some embodiments, each His is, independently, an amino acid selected from the group consisting of E and S. In some embodiments, each H16 is, independently, absent. In some embodiments, each H16 is, independently, an amino acid selected from the group consisting of I, L, V, M, A, and T. In some embodiments, each H17 is, independently, absent. In some embodiments, each H17 is, independently, an amino acid selected from the group consisting of T, G, V, W, and A. In some embodiments, each H17 is, independently, an amino acid selected from the group consisting of T, G, and V. In some embodiments, each H18 is, independently, absent. In some embodiments, each H18 is, independently, an amino acid selected from the group consisting of D, E, S, T, K, and G. In some embodiments, each H19 is, independently, absent. In some embodiments, each H19 is, independently, an amino acid selected from the group consisting of Y, F, and L. In some embodiments, each H20 is, independently, absent. In some embodiments, each H20 is, independently, an amino acid selected from the group consisting of N, Q, S, T, R, and F. In some embodiments, each H21 is, independently, absent. In some embodiments, each H21 is, independently, an amino acid selected from the group consisting of S, K, T, A, Y, M, and F. In some embodiments, each H21 is, independently, an amino acid selected from the group consisting of S and K. In some embodiments, each H22 is, independently, absent. In some embodiments, each H22 is, independently, an amino acid selected from the group consisting of T, Q, S, D, C, V, and L. In some embodiments, each H23 is, independently, absent. In some embodiments, each H23 is, independently, an amino acid selected from the group consisting of G, S, K, N, H, D, W, and L. In some embodiments, each H24 is, independently, absent. In some embodiments, each H24 is, independently, an amino acid selected from the group consisting of I, L, V, P, N, and E. In some embodiments, each H25 is, independently, absent. In some embodiments, each H25 is, independently, an amino acid selected from the group consisting of A, T, G, R, Y, L, F, and E. In some embodiments, each H25 is, independently, A. In some embodiments, each H26 is, independently, absent. In some embodiments, each H26 is, independently, an amino acid selected from the group consisting of V, I, F, M, L, A, and T. In some embodiments, each H26 is, independently, an amino acid selected from the group consisting of V, I, and F. In some embodiments, each H27 is, independently, absent. In some embodiments, each H27 is, independently, an amino acid selected from the group consisting of D, E, Q, N, S, A, and I. In some embodiments, each H28 is, independently, absent. In some embodiments, each H28 is, independently, an amino acid selected from the group consisting of P, S, R, T, N, G, D, K, and A. In some embodiments, each H28 is, independently, an amino acid selected from the group consisting of P, S, and R. In some embodiments, each H29 is, independently, absent. In some embodiments, each H29 is, independently, an amino acid selected from the group consisting of E, D, T, A, Y, M, V, I, F, and L. In some embodiments, each H30 is, independently, absent. In some embodiments, each H30 is, independently, an amino acid selected from the group consisting of T, Q, A, E, F, and S. In some embodiments, each H30 is, independently, T. In some embodiments, each H31 is, independently, absent. In some embodiments, each H31 is, independently, an amino acid selected from the group consisting of F, W, V, M, S, G, and R. In some embodiments, each H32 is, independently, absent. In some embodiments, each H32 is, independently, an amino acid selected from the group consisting of H, S, E, G, and T. In some embodiments, each H33 is, independently, absent. In some embodiments, each H33 is, independently, an amino acid selected from the group consisting of A, T, G, R, Y, L, F, and E. In some embodiments, each H33 is, independently, A. In some embodiments, each H34 is, independently, absent. In some embodiments, each H34 is, independently, an amino acid selected from the group consisting of S, K, T, A, Y, M, and F. In some embodiments, each H34 is, independently, an amino acid selected from the group consisting of S and K. In some embodiments, each H35 is, independently, absent. In some embodiments, each H35 is, independently, an amino acid selected from the group consisting of R, K, S, and Q. In some embodiments, each H36 is, independently, absent. In some embodiments, each H36 is, independently, an amino acid selected from the group consisting of H, R, S, T, A, V, W, and L. In some embodiments, H37 is an amino acid selected from the group consisting of K, Q, D, A, and I. In some embodiments, H38 is an amino acid selected from the group consisting of R, K, T, and F. In some embodiments, H39 is an amino acid selected from the group consisting of D, N, S, T, K, A, Y, and L. In some embodiments, H40 is an amino acid selected from the group consisting of V, I, F, M, L, A, and T. In some embodiments, H40 is an amino acid selected from the group consisting of V, I, and F.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 22, 23, and 24.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence represented by:
(I1)m-(I2)m-(I3)m-(I4)m-(I5)m-(I6)m-(I7)x-(I8)m-(I9)m-(I10)m-(I11)x- (I12)m-(I13)x-(I14)x-(I15)m-(I16)x-(I17)m-I18-I19-I20-I21-I22-I23 (Formula VIII)
wherein each m is, independently, 0, 1, or 2 and each x is, independently, 0, 1, 2, 3, or 4. Table 12 below describes the various amino acids that may be used at each position, with preferable amino acids underlined.
S, Q, E, A, I, G, V, R, T, Y
F, L, W, A, T, M, Y, C
I, L, V
G, S, N, E, D, A, K, H, C,
I, L, V, A, T, S
1-1.3
E, Q, S, T, R, K, A, L, D, F
T, S, Q, F, A, G, V, I, L
I, L, V, N, A, T, S
R, K, Q, A
In some embodiments, amino acid positions I1-I6, I8, I9, I12, I15, and I17 may be omitted or repeated up to 1 extra time (i.e., be included 0 to 2 times), each repeat being independently selected from the indicated amino acids. In some embodiments, amino acid positions I7, I11, I13, I14, and I16 may be omitted or repeated up to 3 extra time (i.e., be included 0 to 4 times), each repeat being independently selected from the indicated amino acids. Further, it is to be understood that the omission or repetition of any amino acid positions 1-9 and 11-17 is independent of the omission or repetition of any amino acid at an alternate position. In some embodiments, the minimum length of a sequence generated using Formula VIII is 17 amino acids.
In some embodiments, each I1 is, independently, absent. In some embodiments, each I1 is, independently, an amino acid selected from the group consisting of S, Q, E, A, I, G, V, R, T, and Y. In some embodiments, each I1 is, independently, an amino acid selected from the group consisting of A, Q, and E. In some embodiments, each I2 is, independently, absent. In some embodiments, each I2 is, independently, an amino acid selected from the group consisting of T, S, E, R, P, V, I, and F. In some embodiments, each I3 is, independently, absent. In some embodiments, each I3 is, independently, L. In some embodiments, each I4 is, independently, absent. In some embodiments, each I4 is, independently, an amino acid selected from the group consisting of T, N, K, and M. In some embodiments, each I5 is, independently, absent. In some embodiments, each I5 is, independently, an amino acid selected from the group consisting of P, A, and D. In some embodiments, each I6 is, independently, absent. In some embodiments, each I6 is, independently, an amino acid selected from the group consisting of S, Q, E, A, I, G, V, R, T, and Y. In some embodiments, each I6 is, independently, an amino acid selected from the group consisting of A, Q, and E. In some embodiments, each I7 is, independently, absent. In some embodiments, each I7 is, independently, an amino acid selected from the group consisting of T, S, K, H, Y, V, and F. In some embodiments, each I8 is, independently, absent. In some embodiments, each I8 is, independently, an amino acid selected from the group consisting of F, L, W, A, T, M, Y, and C. In some embodiments, each I8 is, independently, an amino acid selected from the group consisting of F, L, W, A, and T. In some embodiments, each I9 is, independently, absent. In some embodiments, each I9 is, independently, an amino acid selected from the group consisting of I, L, and V. In some embodiments, each I10 is, independently, absent. In some embodiments, each I10 is, independently, an amino acid selected from the group consisting of G, S, N, E, D, A, K, H, C, P, and F. In some embodiments, each I10 is, independently, an amino acid selected from the group consisting of G and S. In some embodiments, each I11 is, independently, absent. In some embodiments, each I11 is, independently, an amino acid selected from the group consisting of I, L, V, A, T, and S. In some embodiments, each I12 is, independently, absent. In some embodiments, each I12 is, independently, an amino acid selected from the group consisting of T, N, A, E, and G. In some embodiments, each I13 is, independently, absent. In some embodiments, each I13 is, independently, an amino acid selected from the group consisting of E, Q, S, T, R, K, A, L, D, and F. In some embodiments, each I13 is, independently, E. In some embodiments, each I14 is, independently, absent. In some embodiments, each I14 is, independently, an amino acid selected from the group consisting of T, S, Q, F, A, G, V, I, and L. In some embodiments, each I14 is, independently, an amino acid selected from the group consisting of T and S. In some embodiments, each I15 is, independently, absent. In some embodiments, each I15 is, independently, an amino acid selected from the group consisting of F, L, W, A, T, M, Y, and C. In some embodiments, each I15 is, independently, an amino acid selected from the group consisting of F, L, W, A, and T. In some embodiments, each I16 is, independently, absent. In some embodiments, each I16 is, independently, an amino acid selected from the group consisting of G, S, N, E, D, A, K, H, C, P, and F. In some embodiments, each I16 is, independently, an amino acid selected from the group consisting of G and S. In some embodiments, each I17 is, independently, absent. In some embodiments, each I17 is, independently, an amino acid selected from the group consisting of I, L, V, N, A, T, and S. In some embodiments, each I17 is, independently, an amino acid selected from the group consisting of I, L, and V. In some embodiments, I18 is an amino acid selected from the group consisting of R, K, Q, and A. In some embodiments, I18 is R. In some embodiments, I19 is an amino acid selected from the group consisting of H, R, S, N, T, A, V, and W. In some embodiments, I20 is an amino acid selected from the group consisting of K, N, Q, D, E, A, and I. In some embodiments, I21 is an amino acid selected from the group consisting of R, K, Q, and A. In some embodiments, I21 is R. In some embodiments, I22 is an amino acid selected from the group consisting of D, N, S, A, Y, and L. In some embodiments, I23 is an amino acid selected from the group consisting of V, I, L, F, and A.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence represented by:
(J1)z-(J2)z-(J3)z-(J4)z-(J5)z-(J6)z-(J7)z-(J8)z-(J9)z-(J10)z-(J11)z- (J12)z-(J13)z-(J14)z-(J15)z-(J16)z-(J17)z-(J18)z-(J19)z-(J20)z-(J21)z-J22-J23-J24-J25 (Formula X)
wherein each z is, independently, 0, 1, 2, 3, 4, or 5. Table 13 below describes the various amino acids that may be used at each position, with preferable amino acids underlined.
D, E, N, G, P, H, T, R, K,
D, E, N, G, P, H, T, R, K,
L, V, A, G, E, I, P, R
5-11
R, T, V, I, L
L, V, A, G, E, I, P, R
R, T, V, I, L
1-1.3
In some embodiments, amino acid positions J1-J21 may be omitted or repeated up to 4 extra time (i.e., be included 0 to 5 times), each repeat being independently selected from the indicated amino acids. Further, it is to be understood that the omission or repetition of any amino acid positions J1-J21 is independent of the omission or repetition of any amino acid at an alternate position.
In some embodiments, each J1 is, independently, absent. In some embodiments, each J1 is, independently, an amino acid selected from the group consisting of H, K, G, A, P, F, and L. In some embodiments, each J2 is, independently, absent. In some embodiments, each J2 is, independently, an amino acid selected from the group consisting of D, E, N, G, P, H, T, R, K, and A. In some embodiments, each J2 is, independently, an amino acid selected from the group consisting of D, E, N, G, and P. In some embodiments, each J3 is, independently, absent. In some embodiments, each J3 is, independently, an amino acid selected from the group consisting of G, A, P, V, and L. In some embodiments, each J4 is, independently, absent. In some embodiments, each J4 is, independently, an amino acid selected from the group consisting of F, I, P, A, S, E, D, R, and K. In some embodiments, each J5 is, independently, absent. In some embodiments, each J5 is, independently, an amino acid selected from the group consisting of S, R, T, G, K, E, D, and C. In some embodiments, each J6 is, independently, absent. In some embodiments, each J6 is, independently, an amino acid selected from the group consisting of T, S, A, D, and F. In some embodiments, each J7 is, independently, absent. In some embodiments, each J7 is, independently, an amino acid selected from the group consisting of D, E, N, G, P, H, T, R, K, and A. In some embodiments, each J7 is, independently, an amino acid selected from the group consisting of D, E, N, G, and P. In some embodiments, each J8 is, independently, absent. In some embodiments, each J8 is, independently, an amino acid selected from the group consisting of Y, C, A, W, I, S, E, D, F, L, R, and K. In some embodiments, each J9 is, independently, absent. In some embodiments, each J9 is, independently, an amino acid selected from the group consisting of H, K, N, D, G, T, A, C, Y, V, and L. In some embodiments, each J10 is, independently, absent. In some embodiments, each J10 is, independently, an amino acid selected from the group consisting of L, V, A, G, E, I, P, and R. In some embodiments, each J10 is, independently, an amino acid selected from the group consisting of L, V, A, G, and E. In some embodiments, each J11 is, independently, absent. In some embodiments, each J11 is, independently, an amino acid selected from the group consisting of I, W, V, Y, P, T, N, S, R, and K. In some embodiments, each J12 is, independently, absent. In some embodiments, each J12 is, independently, an amino acid selected from the group consisting of A, G, Q, N, R, Y, E, D, and L. In some embodiments, each J13 is, independently, absent. In some embodiments, each J13 is, independently, an amino acid selected from the group consisting of I, L, W, V, M, Y, P, A, S, and G. In some embodiments, each J14 is, independently, absent. In some embodiments, each J14 is, independently, an amino acid selected from the group consisting of V, C, L, F, A, T, N, G, and R. In some embodiments, each J15 is, independently, absent. In some embodiments, each J15 is, independently, an amino acid selected from the group consisting of G, S, R, K, A, T, H, E, W, L, and F. In some embodiments, each J16 is, independently, absent. In some embodiments, each J16 is, independently, an amino acid selected from the group consisting of D, E, Q, S, H, T, R, G, Y, V, F, and L. In some embodiments, each J17 is, independently, absent. In some embodiments, each J17 is, independently, an amino acid selected from the group consisting of E, S, G, Y, I, and L. In some embodiments, each J18 is, independently, absent. In some embodiments, each J18 is, independently, an amino acid selected from the group consisting of A, S, P, H, and V. In some embodiments, each J19 is, independently, absent. In some embodiments, each J19 is, independently, an amino acid selected from the group consisting of N, E, R, K, and A. In some embodiments, each J20 is, independently, absent. In some embodiments, each J20 is, independently, an amino acid selected from the group consisting of R, T, V, I, and L. In some embodiments, each J20 is, independently, R. In some embodiments, each J21 is, independently, absent. In some embodiments, each J21 is, independently, an amino acid selected from the group consisting of L, V, A, G, E, I, P, and R. In some embodiments, each J21 is, independently, an amino acid selected from the group consisting of L, V, A, G, and E. In some embodiments, each J22 is, independently, absent. In some embodiments, J22 is an amino acid selected from the group consisting of K, R, D, T, M, and W. In some embodiments, J23 is an amino acid selected from the group consisting of R, T, V, I, and L. In some embodiments, J24 is an amino acid selected from the group consisting of S, N, G, E, D, P, and W. In some embodiments, J25 is an amino acid selected from the group consisting of A, T, S, Y, M, V, and L.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 34, 35, 36, 37, and 38.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence represented by:
(K1)b-(K2)b-(K3)b-(K4)b-(K5)b-(K6)b-(K7)b-(K8)b-(K9)b-(K10)b-(K11)b- (K12)b-(K13)b-(K14)b-(K15)b-(K16)b-(K17)b-(K18)b-(K19)b-(K20)b-(K21)b-(K22)b-(K23)b-(K24)b-(K25)b-(K26)b-(K27)b-(K28)b-(K29)b-(K30)b-(K31)b-(K32)b-(K33)b-(K34)b-(K35)b-(K36)b-(K37)b-(K38)b-(K39)b-(K40)b-(K41)b-(K42)b-(K43)b-(K44)b-(K45)b-(K46)b-(K47)b-(K48)b-(K49)b-(K50)b-(K51)b-(K52)b-(K53)b-(K54)b-(K55)b-(K56)b-(K57)b-(K58)b-(K59)b-(K60)b-(K61)b-(K62)b-(K63)b-(K64)b-(K65)b-(K66)b-(K67)b-(K68)b-(K69)b-(K70)b-(K71)b-(K72)b-(K73)b-(K74)b-(K75)b-(K76)b-(K77)b-(K78)b-(K79)b-(K80)b-(K81)b-(K82)b-(K83)b-(K84)b-(K85)b-(K86)b-(K87)b-(K88)b-K89-K89-K89-K89-K89 (Formula XI)
wherein each b is, independently, 0, 1, 2, or 3. Table 14 below describes the various amino acids that may be used at each position, with preferable amino acids underlined.
G, S, N, T, Q, D, P, L, F,
E, A, V, Q, G, Y, M, I, L
S, Q, R, T, D, G, E, A, K
A, T, Q, G, R, K, D, L, F,
G, S, N, T, Q, D, P, L, F,
G, S, T, E, P, W, R, N, Q
D, Q, S, G, V, E, N, H, R,
R, K, S, Q, T, Y, N, V, I,
5-11
R, K, S, Q, T, Y, N, V, I,
5-11
D, T, G, A, Y, N, S, C, P,
P, A, E, L, T, Q, S, G, K,
R, S, N, A, P, Y, V, I, F,
E, S, T, V, I, H, A, P, F, L
A, T, Q, P, R, V, N, E, L
S, Q, R, T, D, G, E, A, K
G, S, N, T, Q, D, P, L, F,
L, T, F, V, P, A, K, I
G, S, K, N, T, Q, D, A, P,
A, S, V, G, Q, R, E, D, L,
G, S, T, E, P, W, R, N, Q
S, H, Y, F, N, Q, R, T, G,
K, S, G, D, T, L, R, E, Y,
A, T, Q, G, R, K, D, L, F,
E, S, T, V, I, H, A, P, F, L
A, G, S, Q, R, E, D, V, L,
E, A, V, Q, G, Y, M, I, L
G, S, T, E, P, W, R, N, Q
A, G, P, M, N, V, S
T, Q, E, N, S, A, Y, V,
D, Q, S, G, V, E, N, H, R,
G, S, K, A, C, F, N, T, Q,
P, A, E, L, T, Q, S, G, K,
L, T, F, V, P, A, K, I
D, T, G, A, Y, N, S, C, P,
A, S, V, G, Q, R, E, D, L,
A, T, Q, S, R, V, L, G, H,
T, Q, E, N, S, A, Y, V,
R, S, N, A, P, Y, V, I, F,
R, S, G, N, Q, A, Y, W
A, G, P, M, N, V, S
In some embodiments, amino acid positions K1-K88 may be omitted or repeated up to 2 extra time (i.e., be included 0 to 3 times), each repeat being independently selected from the indicated amino acids. Further, it is to be understood that the omission or repetition of any amino acid positions K1-K88 is independent of the omission or repetition of any amino acid at an alternate position.
In some embodiments, each K1 is, independently, absent. In some embodiments, each K1 is, independently, an amino acid selected from the group consisting of S, G, D, A, C, P, and Y. In some embodiments, each K2 is, independently, absent. In some embodiments, each K2 is, independently, an amino acid selected from the group consisting of Q, S, E, T, R, K, G, A, Y, M, V, and I. In some embodiments, each K3 is, independently, absent. In some embodiments, each K3 is, independently, an amino acid selected from the group consisting of G, S, N, T, Q, D, P, L, F, V, K, A, and C. In some embodiments, each K3 is, independently, G. In some embodiments, each K4 is, independently, absent. In some embodiments, each K4 is, independently, an amino acid selected from the group consisting of R, G, N, D, A, P, Y, and L. In some embodiments, each K5 is, independently, absent. In some embodiments, each K5 is, independently, an amino acid selected from the group consisting of E, A, V, Q, G, Y, M, I, and L. In some embodiments, each K5 is, independently, an amino acid selected from the group consisting of E, A, and V. In some embodiments, each K6 is, independently, absent. In some embodiments, each K6 is, independently, an amino acid selected from the group consisting of S, Q, R, T, D, G, E, A, and K. In some embodiments, each K6 is, independently, an amino acid selected from the group consisting of S, Q, R, T, and D. In some embodiments, each K7 is, independently, absent. In some embodiments, each K7 is, independently, an amino acid selected from the group consisting of N, Q, R, H, K, A, I, F, and L. In some embodiments, each K8 is, independently, absent. In some embodiments, each K8 is, independently, an amino acid selected from the group consisting of A, T, Q, G, R, K, D, L, F, C, V, S, and H. In some embodiments, each K8 is, independently, A. In some embodiments, each K9 is, independently, absent. In some embodiments, each K9 is, independently, an amino acid selected from the group consisting of G, S, N, T, Q, D, P, L, F, V, K, A, and C. In some embodiments, each K9 is, independently, G. In some embodiments, each K10 is, independently, absent. In some embodiments, each K10 is, independently, an amino acid selected from the group consisting of K, H, E, A, Y, L, and F. In some embodiments, each K11 is, independently, absent. In some embodiments, each K11 is, independently, an amino acid selected from the group consisting of S, T, K, E, A, C, W, F, and L. In some embodiments, each K12 is, independently, absent. In some embodiments, each K12 is, independently, an amino acid selected from the group consisting of K, R, H, S, Q, D, E, and A. In some embodiments, each K13 is, independently, absent. In some embodiments, each K13 is, independently, an amino acid selected from the group consisting of G, S, T, E, P, W, R, N, and Q. In some embodiments, each K13 is, independently, G. In some embodiments, each K14 is, independently, absent. In some embodiments, each K14 is, independently, an amino acid selected from the group consisting of D, Q, S, G, V, E, N, H, R, P, and F. In some embodiments, each K14 is, independently, an amino acid selected from the group consisting of D, Q, S, G, and V. In some embodiments, each K15 is, independently, absent. In some embodiments, each K15 is, independently, an amino acid selected from the group consisting of C, A, M, V, S, E, G, I, F, and L. In some embodiments, each K16 is, independently, absent. In some embodiments, each K16 is, independently, an amino acid selected from the group consisting of R, K, S, Q, T, Y, N, V, I, L, and C. In some embodiments, each K16 is, independently, an amino acid selected from the group consisting of R, K, S, Q, T, and Y. In some embodiments, each K17 is, independently, absent. In some embodiments, each K17 is, independently, an amino acid selected from the group consisting of A, G, S, Q, Y, E, D, H, and I. In some embodiments, each K18 is, independently, absent. In some embodiments, each K18 is, independently, an amino acid selected from the group consisting of R, K, S, Q, T, Y, N, V, I, L, and C. In some embodiments, each K18 is, independently, an amino acid selected from the group consisting of R, K, S, Q, T, and Y. In some embodiments, each K19 is, independently, absent. In some embodiments, each K19 is, independently, an amino acid selected from the group consisting of E, D, T, H, K, G, P, V, and L. In some embodiments, each K20 is, independently, absent. In some embodiments, each K20 is, independently, an amino acid selected from the group consisting of F, L, I, V, M, T, G, and R. In some embodiments, each K21 is, independently, absent. In some embodiments, each K21 is, independently, an amino acid selected from the group consisting of E, D, S, G, A, C, and P. In some embodiments, each K22 is, independently, absent. In some embodiments, each K22 is, independently, an amino acid selected from the group consisting of D, T, G, A, Y, N, S, C, P, W, and I. In some embodiments, each K22 is, independently, an amino acid selected from the group consisting of D, T, G, A, and Y. In some embodiments, each K23 is, independently, absent. In some embodiments, each K23 is, independently, an amino acid selected from the group consisting of G, S, N, E, D, Y, and L. In some embodiments, each K24 is, independently, absent. In some embodiments, each K24 is, independently, an amino acid selected from the group consisting of T, S, E, G, P, and I. In some embodiments, each K25 is, independently, absent. In some embodiments, each K25 is, independently, an amino acid selected from the group consisting of K, S, G, T, and L. In some embodiments, each K26 is, independently, absent. In some embodiments, each K26 is, independently, an amino acid selected from the group consisting of S, G, K, E, D, P, and F. In some embodiments, each K27 is, independently, absent. In some embodiments, each K27 is, independently, an amino acid selected from the group consisting of P, A, E, L, T, Q, S, G, K, Y, F, C, V, W, and R. In some embodiments, each K27 is, independently, an amino acid selected from the group consisting of P and A. In some embodiments, each K28 is, independently, absent. In some embodiments, each K28 is, independently, an amino acid selected from the group consisting of E, D, Q, S, T, P, and L. In some embodiments, each K29 is, independently, absent. In some embodiments, each K29 is, independently, an amino acid selected from the group consisting of A, T, S, E, V, W, and I. In some embodiments, each K30 is, independently, absent. In some embodiments, each K30 is, independently, an amino acid selected from the group consisting of K, H, S, G, N, Q, P, and Y. In some embodiments, each K31 is, independently, absent. In some embodiments, each K31 is, independently, an amino acid selected from the group consisting of L, F, V, P, A, N, G, and H. In some embodiments, each K32 is, independently, absent. In some embodiments, each K32 is, independently, an amino acid selected from the group consisting of A, G, N, P, R, E, and K. In some embodiments, each K33 is, independently, absent. In some embodiments, each K33 is, independently, an amino acid selected from the group consisting of R, S, N, A, P, Y, V, I, F, and G. In some embodiments, each K33 is, independently, an amino acid selected from the group consisting of R and S. In some embodiments, each K34 is, independently, absent. In some embodiments, each K34 is, independently, an amino acid selected from the group consisting of E, S, T, V, I, H, A, P, F, and L. In some embodiments, each K34 is, independently, an amino acid selected from the group comprising E, S, T, V, and I. In some embodiments, each K35 is, independently, absent. In some embodiments, each K35 is, independently, an amino acid selected from the group consisting of A, T, Q, P, R, V, N, E, and L. In some embodiments, each K35 is, independently, an amino acid selected from the group consisting of A, T, Q, P, and R. In some embodiments, each K36 is, independently, absent. In some embodiments, each K36 is, independently, an amino acid selected from the group consisting of R, K, H, G, Q, D, T, Y, and F. In some embodiments, each K37 is, independently, absent. In some embodiments, each K37 is, independently, an amino acid selected from the group consisting of D, E, N, T, C, Y, V, I, and L. In some embodiments, each K38 is, independently, absent. In some embodiments, each K38 is, independently, an amino acid selected from the group consisting of S, Q, R, T, D, G, E, A, and K. In some embodiments, each K38 is, independently, an amino acid selected from the group consisting of S, Q, R, T, and D. In some embodiments, each K39 is, independently, absent. In some embodiments, each K39 is, independently, an amino acid selected from the group consisting of K, S, G, Q, D, E, A, M, I, and L. In some embodiments, each K40 is, independently, absent. In some embodiments, each K40 is, independently, an amino acid selected from the group consisting of H, K, S, D, E, T, P, and L. In some embodiments, each K41 is, independently, absent. In some embodiments, each K41 is, independently, an amino acid selected from the group consisting of A, T, S, N, P, V, L, and F. In some embodiments, each K42 is, independently, absent. In some embodiments, each K42 is, independently, an amino acid selected from the group consisting of K, D, M, V, I, L, and F. In some embodiments, each K43 is, independently, absent. In some embodiments, each K43 is, independently, an amino acid selected from the group consisting of G, S, N, T, Q, D, P, L, F, V, K, A, and C. In some embodiments, each K43 is, independently, G. In some embodiments, each K44 is, independently, absent. In some embodiments, each K44 is, independently, an amino acid selected from the group consisting of L, T, F, V, P, A, K, and I. In some embodiments, each K44 is, independently, an amino acid selected from the group consisting of L and T. In some embodiments, each K45 is, independently, absent. In some embodiments, each K45 is, independently, an amino acid selected from the group consisting of G, S, K, N, T, Q, D, A, P, L, F, and V. In some embodiments, each K45 is, independently, G. In some embodiments, each K46 is, independently, absent. In some embodiments, each K46 is, independently, an amino acid selected from the group consisting of L, F, Q, S, G, and D. In some embodiments, each K47 is, independently, absent. In some embodiments, each K47 is, independently, an amino acid selected from the group consisting of S, R, E, A, P, V, W, and L. In some embodiments, each K48 is, independently, absent. In some embodiments, each K48 is, independently, an amino acid selected from the group consisting of A, S, V, G, Q, R, E, D, L, T, K, F, C, and H. In some embodiments, each K48 is, independently, A. In some embodiments, each K49 is, independently, absent. In some embodiments, each K49 is, independently, an amino acid selected from the group consisting of E, S, T, R, G, A, P, and L. In some embodiments, each K50 is, independently, absent. In some embodiments, each K50 is, independently, an amino acid selected from the group consisting of S, N, R, A, P, and Y. In some embodiments, each K51 is, independently, absent. In some embodiments, each K51 is, independently, an amino acid selected from the group consisting of G, A, T, H, M, V, L, and F. In some embodiments, each K52 is, independently, absent. In some embodiments, each K52 is, independently, an amino acid selected from the group consisting of S, T, H, A, C, M, and L. In some embodiments, each K53 is, independently, absent. In some embodiments, each K53 is, independently, an amino acid selected from the group consisting of G, S, T, E, P, W, R, N, and Q. In some embodiments, each K53 is, independently, G. In some embodiments, each K54 is, independently, absent. In some embodiments, each K54 is, independently, an amino acid selected from the group consisting of S, H, Y, F, N, Q, R, T, G, and K. In some embodiments, each K54 is, independently, S. In some embodiments, each K55 is, independently, absent. In some embodiments, each K55 is, independently, an amino acid selected from the group consisting of A, T, Q, E, M, V, I, L, and F. In some embodiments, each K56 is, independently, absent. In some embodiments, each K56 is, independently, an amino acid selected from the group consisting of S, N, E, A, P, F, and L. In some embodiments, each K57 is, independently, absent. In some embodiments, each K57 is, independently, an amino acid selected from the group consisting of D, S, R, K, A, V, W, I, and F. In some embodiments, each K58 is, independently, absent. In some embodiments, each K58 is, independently, an amino acid selected from the group consisting of K, S, G, D, T, L, R, E, Y, and N. In some embodiments, each K58 is, independently, an amino acid selected from the group consisting of K, S, G, D, T, and L. In some embodiments, each K59 is, independently, absent. In some embodiments, each K59 is, independently, an amino acid selected from the group consisting of S, R, G, A, V, and F. In some embodiments, each K60 is, independently, absent. In some embodiments, each K60 is, independently, an amino acid selected from the group consisting of A, T, Q, G, R, K, D, L, F, C, V, S, and H. In some embodiments, each K60 is, independently, A. In some embodiments, each K61 is, independently, absent. In some embodiments, each K61 is, independently, an amino acid selected from the group consisting of R, S, G, N, E, T, A, and V. In some embodiments, each K62 is, independently, absent. In some embodiments, each K62 is, independently, an amino acid selected from the group consisting of E, S, T, V, I, H, A, P, F, and L. In some embodiments, each K63 is, independently, absent. In some embodiments, each K63 is, independently, an amino acid selected from the group consisting of A, G, S, Q, R, E, D, V, L, T, K, F, C, and H. In some embodiments, each K63 is, independently, A. In some embodiments, each K64 is, independently, absent. In some embodiments, each K64 is, independently, an amino acid selected from the group consisting of E, A, V, Q, G, Y, M, I, and L. In some embodiments, each K64 is, independently, an amino acid selected from the group consisting of E, A, and V. In some embodiments, each K65 is, independently, absent. In some embodiments, each K65 is, independently, an amino acid selected from the group consisting of G, S, T, E, P, W, R, N, and Q. In some embodiments, each K65 is, independently, G. In some embodiments, each K66 is, independently, absent. In some embodiments, each K66 is, independently, an amino acid selected from the group consisting of A, G, P, M, N, V, and S. In some embodiments, each K66 is, independently, an amino acid selected from the group consisting of A, G, P, and M. In some embodiments, each K67 is, independently, absent. In some embodiments, each K67 is, independently, an amino acid selected from the group consisting of T, Q, E, N, S, A, Y, V, W, and F. In some embodiments, each K67 is, independently, an amino acid selected from the group consisting of T, Q, and E. In some embodiments, each K68 is, independently, absent. In some embodiments, each K68 is, independently, an amino acid selected from the group consisting of I, V, P, and A. In some embodiments, each K69 is, independently, absent. In some embodiments, each K69 is, independently, an amino acid selected from the group consisting of D, Q, S, G, V, E, N, H, R, P, and F. In some embodiments, each K69 is, independently, an amino acid selected from the group consisting of D, Q, S, G, and V. In some embodiments, each K70 is, independently, absent. In some embodiments, each K70 is, independently, an amino acid selected from the group consisting of G, S, R, N, T, Y, L, and F. In some embodiments, each K71 is, independently, absent. In some embodiments, each K71 is, independently, an amino acid selected from the group consisting of E, D, N, S, T, H, and Y. In some embodiments, each K72 is, independently, absent. In some embodiments, each K72 is, independently, an amino acid selected from the group consisting of L, I, W, V, A, T, S, E, R, and K. In some embodiments, each K73 is, independently, absent. In some embodiments, each K73 is, independently, an amino acid selected from the group consisting of G, S, K, A, C, F, N, T, Q, D, P, L, and V. In some embodiments, each K73 is, independently, G. In some embodiments, each K74 is, independently, absent. In some embodiments, each K74 is, independently, an amino acid selected from the group consisting of A, S, N, P, K, V, I, and L. In some embodiments, each K75 is, independently, absent. In some embodiments, each K75 is, independently, an amino acid selected from the group consisting of P, A, E, L, T, Q, S, G, K, Y, F, C, V, W, and R. In some embodiments, each K75 is, independently, an amino acid selected from the group consisting of P and A. In some embodiments, each K76 is, independently, absent. In some embodiments, each K76 is, independently, an amino acid selected from the group consisting of L, T, F, V, P, A, K, and I. In some embodiments, each K76 is, independently, an amino acid selected from the group consisting of L and T. In some embodiments, each K77 is, independently, absent. In some embodiments, each K77 is, independently, an amino acid selected from the group consisting of M, V, Y, L, A, N, E, and H. In some embodiments, each K78 is, independently, absent. In some embodiments, each K78 is, independently, an amino acid selected from the group consisting of D, T, G, A, Y, N, S, C, P, W, and I. In some embodiments, each K78 is, independently, an amino acid selected from the group consisting of D, T, G, A, and Y. In some embodiments, each K79 is, independently, absent. In some embodiments, each K79 is, independently, an amino acid selected from the group consisting of A, S, V, G, Q, R, E, D, L, T, K, F, C, and H. In some embodiments, each K79 is, independently, A. In some embodiments, each K80 is, independently, absent. In some embodiments, each K80 is, independently, an amino acid selected from the group consisting of K, R, S, A, P, V, I, and L. In some embodiments, each K81 is, independently, absent. In some embodiments, each K81 is, independently, an amino acid selected from the group consisting of F, L, V, A, T, S, E, D, R, and K. In some embodiments, each K82 is, independently, absent. In some embodiments, each K82 is, independently, an amino acid selected from the group consisting of L, F, M, A, N, G, and E. In some embodiments, each K83 is, independently, absent. In some embodiments, each K83 is, independently, an amino acid selected from the group consisting of D, S, H, A, V, I, F, and L. In some embodiments, each K84 is, independently, absent. In some embodiments, each K84 is, independently, an amino acid selected from the group consisting of A, T, Q, S, R, V, L, G, H, F, K, D, and C. In some embodiments, each K84 is, independently, A. In some embodiments, each K85 is, independently, absent. In some embodiments, each K85 is, independently, an amino acid selected from the group consisting of T, Q, E, N, S, A, Y, V, W, and F. In some embodiments, each K85 is, independently, an amino acid selected from the group consisting of T, Q, and E. In some embodiments, each K86 is, independently, absent. In some embodiments, each K86 is, independently, an amino acid selected from the group consisting of A, P, R, Y, K, D, M, L, and F. In some embodiments, each K87 is, independently, absent. In some embodiments, each K87 is, independently, an amino acid selected from the group consisting of N, S, D, T, A, P, and L. In some embodiments, each K88 is, independently, absent. In some embodiments, each K88 is, independently, an amino acid selected from the group consisting of R, S, N, A, P, Y, V, I, F, and G. In some embodiments, each K88 is, independently, an amino acid selected from the group consisting of R and S. In some embodiments, K89 is an amino acid selected from the group consisting of K, R, H, G, E, T, Y, and I. In some embodiments, K90 is an amino acid selected from the group consisting of R, S, G, N, Q, A, Y, and W. In some embodiments, K90 is R. In some embodiments, K91 is an amino acid selected from the group consisting of V, I, and F. In some embodiments, K92 is an amino acid selected from the group consisting of A, G, P, M, N, V, and S. In some embodiments, K92 is an amino acid selected from the group consisting of A, G, P, and M. In some embodiments, K93 is an amino acid selected from the groups consisting of E, D, Q, S, R, K, M, and L.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence represented by:
(M1)b-(M2)b-(M3)b-(M4)b-(M5)b-(M6)b-(M7)b-(M8)b-(M9)b-(M10)b-(M11)b- (M12)b-(M13)b-(M14)b-(M15)b-(M16)b-(M17)b-(M18)b-(M19)b-(M20)b-(M21)b-(M22)b-(M23)b-(M24)b-(M25)b-(M26)b-(M27)b-(M28)b-(M29)b-(M30)b-(M31)b-(M32)b-(M33)b-(M34)b-(M35)b-(M36)b-(M37)b-(M38)b-(M39)b-(M40)b-(M41)b-(M42)b-(M43)b-(M44)b-(M45)b-(M46)b-(M47)b-(M48)b-(M49)b-(M50)b-(M51)b-(M52)b-(M53)b-(M54)b-(M55)b-(M56)b-(M57)b-(M58)b-(M59)b-(M60)b-(M61)b-(M62)b-(M63)b-(M64)b-(M65)b-(M66)b-(M67)c-(M68)c-(M69)c-(M70)c (Formula XIV)
wherein each b is, independently, 0, 1, 2, or 3, and each c is, independently, 1 or 2. Table 15 below describes the various amino acids that may be used at each position, with preferable amino acids underlined.
A, T, C, S, Y, E, H, V, W, I,
S, T, A, N, R, G, E, P, V, F,
G, S, R, A, T, Q, E, D, C,
R, H, N, Q, E, A, Y, M, V,
P, Y, A, T, Q, S, G, D, R,
T, Q, N, S, A, E, G, D, H,
A, G, S, Q, N, K, D, T, C,
T, Q, N, S, A, G, C, R, K,
G, S, H, P, R, A, T, Q, E, D,
Q, E, and W
V, I, L, F, C, A, and T
S, G, A, N, Q, R, T, K, E,
T, Q, N, S, D, P, F, A, E, G,
L, F, I, V, M, Y, A, T, Q, N,
S, P, V, E, T, A, F, L, N, R,
T, S, A, E, G, C, R, P, Y,
D, E, Q, T, K, P, F, N, S, G,
G, S, H, P, R, D, N, A, T,
T, P, F, S, A, E, G, C, R, Y,
L, F, I, V, Y, A, T, Q, S, D,
F, L, W, Y, and P
P, K, Y, A, T, Q, S, G, D,
T, P, F, S, A, E, G, C, R, Y,
S, T, A, N, R, G, E, P, V, F,
F, W, Y, and P
T, P, F, Q, N, S, A, E, G, D,
D, E, Q, N, S, T, R, K, G,
T, Q, N, S, A, G, C, R, K,
S, T, E, A, P, V, F, L, N, R,
D, Q, N, H, K, G, C, and Y
F, L, W, Y, and P
S, T, E, A, P, V, F, L, N, R,
A, G, S, Q, N, K, D, T, C,
T, A, V, I, P, F, Q, N, S, E,
G, S, R, N, H, D, P, A, T,
T, Q, S, A, E, D, K, H, P,
I, L, W, V, and M
14-34
A, G, S, Q, N, K, D, C, P,
S, T, E, P, V, A, F, L, N, R,
T, S, A, D, P, M, Q, E, K,
L, F, I, V, Y, A, T, Q, S, D,
P, Y, A, T, Q, S, N, W, G, I,
E, D, L, K, and H
S, E, P, V, T, A, F, L, N, R,
N, Q, S, E, D, T, H, K, G,
A, P, W, and F
V, I, L, F, C, A, and T
A, T, S, N, R, Y, K, D, H,
I, L, and V
14-25
S, P, V, E, T, A, F, L, N, R,
F, V, A, T, Q, N, S, E, G,
D, and H
L, F, I, V, Y, A, T, Q, S, D,
G, S, R, H, D, P, N, A, T,
T, N, S, G, C, R, H, A, D,
L, L, W, V, and M
14-34
P, K, Y, A, T, Q, S, G, D,
D, E, Q, N, S, K, G, A, Y,
L, F, I, V, Y, P, A, T, Q, N,
S, P, V, E, T, A, F, L, N, R,
P, M, V, I, L, and F
N, Q, S, E, D, T, R, K, G,
A, and Y
G, S, H, P, R, D, N, A, T,
S, P, V, T, A, R, K, E, H, C,
P, K, A, Y, T, Q, S, G, D,
A, G, S, N, E, K, D, H, M,
D, E, Q, T, K, P, F, N, S, G,
L, V, F, I, Y, P, A, T, Q, N,
S, N, R, T, G, K, E, H, D,
K, R, H, S, G, N, Q, D, E,
R, K, H, S, G, N, Q, D, E,
S, A, N, Q, R, T, G, K, E,
T, Q, N, S, A, E, G, D, C,
In some embodiments, amino acid positions M1-M66 may be omitted or repeated up to 2 extra time (i.e., be included 0 to 3 times), each repeat being independently selected from the indicated amino acids. It is to be understood that the omission or repetition of any amino acid positions M1-M66 is independent of the omission or repetition of any amino acid at an alternate position. In some embodiments, amino acid positions M67-M70 may be repeated up to 1 extra time (i.e., be included 1 to 2 times), each repeat being independently selected from the indicated amino acids. It is to be understood that the repetition of any amino acid positions M67-M70 is independent of the repetition of any amino acid at an alternate position.
In some embodiments, each M1 is, independently, absent. In some embodiments, each M1 is, independently, an amino acid selected from the group consisting of A, T, C, S, Y, E, H, V, W, I, L, F, G, Q, N, P, R, K, D, and M. In some embodiments, each M1 is, independently, A. In some embodiments, each M2 is, independently, absent. In some embodiments, each M2 is, independently, an amino acid selected from the group consisting of S, T, A, N, R, G, E, P, V, F, L, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M2 is, independently, S. In some embodiments, each M3 is, independently, absent. In some embodiments, each M3 is, independently, an amino acid selected from the group consisting of G, S, R, A, T, Q, E, D, C, Y, I, L, and N. In some embodiments, each M3 is, independently, G. In some embodiments, each M4 is, independently, absent. In some embodiments, each M4 is, independently, an amino acid selected from the group consisting of R, H, N, Q, E, A, Y, M, V, W, F, and L. In some embodiments, each M4 is, independently, R. In some embodiments, each M5 is, independently, absent. In some embodiments, each M5 is, independently, an amino acid selected from the group consisting of P, Y, A, T, Q, S, G, D, R, K, C, V, I, L, and H. In some embodiments, each M5 is, independently, P. In some embodiments, each M6 is, independently, absent. In some embodiments, each M6 is, independently, an amino acid selected from the group consisting of T, Q, N, S, A, E, G, D, H, P, F, L, C, K, V, R, Y, I, M, and W. In some embodiments, each M6 is, independently, T. In some embodiments, each M7 is, independently, absent. In some embodiments, each M7 is, independently, an amino acid selected from the group consisting of A, G, S, Q, N, K, D, T, C, Y, E, H, V, W, I, L, F, P, R, and M. In some embodiments, each M7 is, independently, A. In some embodiments, each M8 is, independently, absent. In some embodiments, each M8 is, independently, an amino acid selected from the group consisting of T, Q, N, S, A, G, C, R, K, P, Y, M, V, I, L, F, E, W, D, and H. In some embodiments, each M8 is, independently, T. In some embodiments, each M9 is, independently, absent. In some embodiments, each M9 is, independently, an amino acid selected from the group consisting of G, S, H, P, R, A, T, Q, E, D, C, Y, V, I, L, N, W, F, K, and M. In some embodiments, each M9 is, independently, G. In some embodiments, each M10 is, independently, absent. In some embodiments, each M10 is, independently, an amino acid selected from the group consisting of Q, E, and W. In some embodiments, each Mn is, independently, absent. In some embodiments, each M11 is, independently, an amino acid selected from the group consisting of V, I, L, F, C, A, and T. In some embodiments, each M11 is, independently, an amino acid selected from the group consisting of V, I, and L. In some embodiments, each M12 is, independently, absent. In some embodiments, each M12 is, independently, an amino acid selected from the group consisting of S, G, A, N, Q, R, T, K, E, H, D, P, I, F, V, C, Y, L, M, and W. In some embodiments, each M12 is, independently, S. In some embodiments, each M13 is, independently, absent. In some embodiments, each M13 is, independently, an amino acid selected from the group consisting of T, Q, N, S, D, P, F, A, E, G, H, L, C, K, V, R, Y, I, M, and W. In some embodiments, each M13 is, independently, T. In some embodiments, each M14 is, independently, absent. In some embodiments, each M14 is, independently, an amino acid selected from the group consisting of L, F, I, V, M, Y, A, T, Q, N, S, D, K, P, E, R, H, G, and C. In some embodiments, each M14 is, independently, L. In some embodiments, each M15 is, independently, absent. In some embodiments, each M15 is, independently, an amino acid selected from the group consisting of S, P, V, E, T, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M15 is, independently, S. In some embodiments, each M16 is, independently, absent. In some embodiments, each M16 is, independently, an amino acid selected from the group consisting of T, S, A, E, G, C, R, P, Y, M, V, W, I, F, L, Q, N, D, H, and K. In some embodiments, each M16 is, independently, T. In some embodiments, each M17 is, independently, absent. In some embodiments, each M17 is, independently, an amino acid selected from the group consisting of D, E, Q, T, K, P, F, N, S, G, A, Y, R, and V. In some embodiments, each M17 is, independently, D. In some embodiments, each M18 is, independently, absent. In some embodiments, each M18 is, independently, an amino acid selected from the group consisting of G, S, H, P, R, D, N, A, T, Q, E, C, Y, V, I, L, W, F, K, and M. In some embodiments, each M18 is, independently, G. In some embodiments, each M19 is, independently, absent. In some embodiments, each M19 is, independently, an amino acid selected from the group consisting of T, P, F, S, A, E, G, C, R, Y, M, V, W, I, L, Q, N, D, H, and K. In some embodiments, each M19 is, independently, T. In some embodiments, each M20 is, independently, absent. In some embodiments, each M20 is, independently, an amino acid selected from the group consisting of L, F, I, V, Y, A, T, Q, S, D, M, N, K, P, E, R, H, G, and C. In some embodiments, each M20 is, independently, L. In some embodiments, each M21 is, independently, absent. In some embodiments, each M21 is, independently, an amino acid selected from the group consisting of F, L, W, Y, and P. In some embodiments, each M21 is, independently, F. In some embodiments, each M22 is, independently, absent. In some embodiments, each M22 is, independently, an amino acid selected from the group consisting of P, K, Y, A, T, Q, S, G, D, R, C, V, I, L, and H. In some embodiments, each M22 is, independently, P. In some embodiments, each M23 is, independently, absent. In some embodiments, each M23 is, independently, an amino acid selected from the group consisting of T, P, F, S, A, E, G, C, R, Y, M, V, W, I, L, Q, N, D, H, and K. In some embodiments, each M23 is, independently, T. In some embodiments, each M24 is, independently, absent. In some embodiments, each M24 is, independently, an amino acid selected from the group consisting of S, T, A, N, R, G, E, P, V, F, L, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M24 is, independently, S. In some embodiments, each M25 is, independently, absent. In some embodiments, each M25 is, independently, an amino acid selected from the group consisting of F, W, Y, and P. In some embodiments, each M25 is, independently, F. In some embodiments, each M26 is, independently, absent. In some embodiments, each M26 is, independently, an amino acid selected from the group consisting of T, P, F, Q, N, S, A, E, G, D, K, Y, C, V, I, L, and H. In some embodiments, each M26 is, independently, T. In some embodiments, each M27 is, independently, absent. In some embodiments, each M27 is, independently, an amino acid selected from the group consisting of D, E, Q, N, S, T, R, K, G, A, Y, P, V, and F. In some embodiments, each M27 is, independently, D. In some embodiments, each M28 is, independently, absent. In some embodiments, each M28 is, independently, an amino acid selected from the group consisting of T, Q, N, S, A, G, C, R, K, P, Y, M, V, I, L, F, E, W, D, and H. In some embodiments, each M28 is, independently, T. In some embodiments, each M29 is, independently, absent. In some embodiments, each M29 is, independently, an amino acid selected from the group consisting of S, T, E, A, P, V, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M29 is, independently, S. In some embodiments, each M30 is, independently, absent. In some embodiments, each M30 is, independently, an amino acid selected from the group consisting of D, Q, N, H, K, G, C, and Y. In some embodiments, each M31 is, independently, absent. In some embodiments, each M31 is, independently, an amino acid selected from the group consisting of F, L, W, Y, and P. In some embodiments, each M31 is, independently, F. In some embodiments, each M32 is, independently, absent. In some embodiments, each M32 is, independently, an amino acid selected from the group consisting of S, T, E, A, P, V, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M32 is, independently, S. In some embodiments, each M33 is, independently, absent. In some embodiments, each M33 is, independently, an amino acid selected from the group consisting of A, G, S, Q, N, K, D, T, C, Y, E, H, V, W, I, L, F, P, R, and M. In some embodiments, each M33 is, independently, A. In some embodiments, each M34 is, independently, absent. In some embodiments, each M34 is, independently, an amino acid selected from the group consisting of T, A, V, I, P, F, Q, N, S, E, G, D, K, Y, C, L, and H. In some embodiments, each M34 is, independently, T. In some embodiments, each M35 is, independently, absent. In some embodiments, each M35 is, independently, an amino acid selected from the group consisting of G, S, R, N, H, D, P, A, T, Q, E, C, Y, V, I, L, W, F, K, and M. In some embodiments, each M35 is, independently, G. In some embodiments, each M36 is, independently, absent. In some embodiments, each M36 is, independently, an amino acid selected from the group consisting of T, Q, S, A, E, D, K, H, P, Y, V, W, I, F, L, N, G, and C. In some embodiments, each M36 is, independently, T. In some embodiments, each M37 is, independently, absent. In some embodiments, each M37 is, independently, an amino acid selected from the group consisting of I, L, W, V, and M. In some embodiments, each M37 is, independently, I. In some embodiments, each M38 is, independently, absent. In some embodiments, each M38 is, independently, an amino acid selected from the group consisting of A, G, S, Q, N, K, D, C, P, R, Y, E, V, W, T, H, M, and F. In some embodiments, each M38 is, independently, A. In some embodiments, each M39 is, independently, absent. In some embodiments, each M39 is, independently, an amino acid selected from the group consisting of S, T, E, P, V, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M39 is, independently, S. In some embodiments, each M40 is, independently, absent. In some embodiments, each M40 is, independently, an amino acid selected from the group consisting of T, S, A, D, P, M, Q, E, K, H, Y, V, W, I, F, L, N, G, and C. In some embodiments, each M40 is, independently, T. In some embodiments, each M41 is, independently, absent. In some embodiments, each M41 is, independently, an amino acid selected from the group consisting of L, F, I, V, Y, A, T, Q, S, D, M, N, K, P, E, R, H, G, and C. In some embodiments, each M41 is, independently, L. In some embodiments, each M42 is, independently, absent. In some embodiments, each M42 is, independently, an amino acid selected from the group consisting of P, Y, A, T, Q, S, N, W, G, I, E, D, L, K, and H. In some embodiments, each M43 is, independently, absent. In some embodiments, each M43 is, independently, an amino acid selected from the group consisting of S, E, P, V, T, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M43 is, independently, S. In some embodiments, each M44 is, independently, absent. In some embodiments, each M44 is, independently, an amino acid selected from the group consisting of N, Q, S, E, D, T, H, K, G, A, P, W, and F. In some embodiments, each M45 is, independently, absent. In some embodiments, each M45 is, independently, an amino acid selected from the group consisting of V, I, L, F, C, A, and T. In some embodiments, each M45 is, independently, an amino acid selected from the group consisting of V, I, and L. In some embodiments, each M46 is, independently, absent. In some embodiments, each M46 is, independently, an amino acid selected from the group consisting of A, T, S, N, R, Y, K, D, H, M, L, F, G, Q, C, P, E, V, and W. In some embodiments, each M46 is, independently, A. In some embodiments, each M47 is, independently, absent. In some embodiments, each M47 is, independently, an amino acid selected from the group consisting of I, L, and V. In some embodiments, each M47 is, independently, I. In some embodiments, each M48 is, independently, absent. In some embodiments, each M48 is, independently, an amino acid selected from the group consisting of S, P, V, E, T, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M48 is, independently, S. In some embodiments, each M49 is, independently, absent. In some embodiments, each M49 is, independently, an amino acid selected from the group consisting of F, V, A, T, Q, N, S, E, G, D, and H. In some embodiments, each M50 is, independently, absent. In some embodiments, each M50 is, independently, an amino acid selected from the group consisting of L, F, I, V, Y, A, T, Q, S, D, M, N, K, P, E, R, H, G, and C. In some embodiments, each M50 is, independently, L. In some embodiments, each M51 is, independently, absent. In some embodiments, each M51 is, independently, an amino acid selected from the group consisting of G, S, R, H, D, P, N, A, T, Q, E, C, Y, V, I, L, W, F, K, and M. In some embodiments, each M51 is, independently, G. In some embodiments, each M52 is, independently, absent. In some embodiments, each M52 is, independently, an amino acid selected from the group consisting of T, N, S, G, C, R, H, A, D, P, M, Q, E, K, Y, V, W, I, F, and L. In some embodiments, each M52 is, independently, T. In some embodiments, each M53 is, independently, absent. In some embodiments, each M53 is, independently, an amino acid selected from the group consisting of I, L, W, V, and M. In some embodiments, each M53 is, independently, I. In some embodiments, each M54 is, independently, absent. In some embodiments, each M54 is, independently, an amino acid selected from the group consisting of P, K, Y, A, T, Q, S, G, D, R, C, V, I, L, and H. In some embodiments, each M54 is, independently, P. In some embodiments, each M55 is, independently, absent. In some embodiments, each M55 is, independently, an amino acid selected from the group consisting of D, E, Q, N, S, K, G, A, Y, P, F, T, R, and V. In some embodiments, each M55 is, independently, D. In some embodiments, each M56 is, independently, absent. In some embodiments, each M56 is, independently, an amino acid selected from the group consisting of L, F, I, V, Y, P, A, T, Q, N, S, G, E, D, K, H, M, C, and R. In some embodiments, each M56 is, independently, L. In some embodiments, each M57 is, independently, absent. In some embodiments, each M57 is, independently, an amino acid selected from the group consisting of S, P, V, E, T, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M57 is, independently, S. In some embodiments, each M58 is, independently, absent. In some embodiments, each M58 is, independently, an amino acid selected from the group consisting of P, M, V, I, L, and F. In some embodiments, each M59 is, independently, absent. In some embodiments, each M59 is, independently, an amino acid selected from the group consisting of N, Q, S, E, D, T, R, K, G, A, and Y. In some embodiments, each M60 is, independently, absent. In some embodiments, each M60 is, independently, an amino acid selected from the group consisting of G, S, H, P, R, D, N, A, T, Q, E, C, Y, V, I, L, W, F, K, and M. In some embodiments, each M60 is, independently, G. In some embodiments, each M61 is, independently, absent. In some embodiments, each M61 is, independently, an amino acid selected from the group consisting of S, P, V, T, A, R, K, E, H, C, Y, I, F, L, N, Q, G, D, M, and W. In some embodiments, each M61 is, independently, S. In some embodiments, each M62 is, independently, absent. In some embodiments, each M62 is, independently, an amino acid selected from the group consisting of P, K, A, Y, T, Q, S, G, D, R, C, V, I, L, and H. In some embodiments, each M62 is, independently, P. In some embodiments, each M63 is, independently, absent. In some embodiments, each M63 is, independently, an amino acid selected from the group consisting of A, G, S, N, E, K, D, H, M, V, W, I, L, F, T, R, Y, Q, C, and P. In some embodiments, each M63 is, independently, A. In some embodiments, each M64 is, independently, absent. In some embodiments, each M64 is, independently, an amino acid selected from the group consisting of D, E, Q, T, K, P, F, N, S, G, A, Y, R, and V. In some embodiments, each M64 is, independently, D. In some embodiments, each M65 is, independently, absent. In some embodiments, each M65 is, independently, an amino acid selected from the group consisting of L, V, F, I, Y, P, A, T, Q, N, S, G, E, D, K, H, M, C, and R. In some embodiments, each M65 is, independently, L. In some embodiments, each M66 is, independently, absent. In some embodiments, each M66 is, independently, an amino acid selected from the group consisting of S, N, R, T, G, K, E, H, D, A, P, V, C, Y, I, F, L, Q, M, and W. In some embodiments, each M66 is, independently, S. In some embodiments, each M67 is, independently, an amino acid selected from the group consisting of K, R, H, S, G, N, Q, D, E, T, A, C, P, Y, M, V, W, I, L, and F. In some embodiments, each M67 is, independently, an amino acid selected from the group consisting of K, R, H, and S. In some embodiments, each M68 is, independently, an amino acid selected from the group consisting of R, K, H, S, G, N, Q, D, E, T, A, C, P, Y, M, V, W, I, L, and F. In some embodiments, each M68 is, independently, an amino acid selected from the group consisting of R, K, H, and S. In some embodiments, each M69 is, independently, an amino acid selected from the group consisting of S, A, N, Q, R, T, G, K, E, H, D, A, C, P, Y, M, V, W, I, F, and L. In some embodiments, each M69 is, independently, an amino acid selected from the group consisting of S, A, N, Q, R, and T. In some embodiments, each M70 is, independently, an amino acid selected from the group consisting of T, Q, N, S, A, E, G, D, C, R, K, H, P, Y, M, V, W, I, F, and L. In some embodiments, each M70 is, independently, an amino acid selected from the group consisting of T, Q, N, S, A, and E.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO. 74.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence represented by:
(N1)b-(N2)b-(N3)b-(N4)b-(N5)b-(N6)b-(N7)b-(N8)b-(N9)b-(N10)b-(N11)b- (N12)b-(N13)b-(N14)b-(N15)b-(N16)b-(N17)b-(N18)b-(N19)b-(N20)b-(N21)b-(N22)b-(N23)b-(N24)b-(N25)b-(N26)b-(N27)b-(N28)b-(N29)b-(N30)b-(N31)b-(N32)b-(N33)b-(N34)b-(N35)b-(N36)b-(N37)b-(N38)b-(N39)b-(N40)b-(N41)b-(N42)b-(N43)b-(N44)b-(N45)b-(N46)b-(N47)b-(N48)b-(N49)b-(N50)b-(N51)b-(N52)b-(N53)b-(N54)b-(N55)b-(N56)b-(N57)b-(N58)b-(N59)b-(N60)b-(N61)b-(N62)b-(N63)b-(N64)b-(N65)b-(N66)b-(N67)c-(N68)c-(N69)c-(N70)c-(N71)c (Formula XV)
wherein each b is, independently, 0, 1, 2, or 3, and each c is, independently, 1 or 2. Table 16 below describes the various amino acids that may be used at each position, with preferable amino acids underlined.
S, N, D, Q, R, T, G, E,
P, A, S, Y, V, T, G, I,
T, S, G, D, C, A, L, N,
S, R, E, A, Q, K, N,
T, Q, N, G, C, M, S,
−1-34
I, V, L, F, W, Y, A, T,
P, V, A, S, N, G, E, L,
and K
A, G, Q, T, S, N, P, R,
F, Y, A, T, N, and R
T, Q, N, R, K, M, S,
A, G, Q, T, S, N, P, R,
S, N, Q, R, T, G, K, E,
L, F, I, W, V, M, Y,
C, A, T, Q, N, S, G, E,
D, and R
V, I, L, A, T, S, G, R,
S, N, Q, T, G, K, E,
T, N, S, A, D, R, P, Y,
S, N, Q, R, K, E, D,
V, A, T, S, G, R, W, I,
T, Q, N, S, A, E, G,
−1-34
S, Q, R, K, E, A, N,
V, W, I, C, L, F, A, T,
T, Q, N, S, A, D, C,
L, F, I, V, P, A, T, Q,
S, G, R, K, H, M, Y,
T, Q, S, A, G, P, Y, I,
S, R, E, A, Q, K, N,
T, N, D, S, A, R, P, Y,
D, N, R, E, Q, S, H, T,
V, A, T, S, G, R, W, I,
T, S, A, D, C, L, N, R,
P, Y, V, A, T, S, G, I,
T, Q, S, A, G, K, H, P,
S, R, E, A, Q, K, N,
E, D, Q, N, S, T, H, R,
G, A, P, F, and L
D, N, R, E, Q, S, H, T,
T, Q, S, A, G, P, Y, I,
G, S, K, A, T, Q, D,
C, P, Y, V, W, I, L,
and F
F, Y, A, T, N, and R
V, A, T, S, G, R, W, I,
L, F, I, W, V, M, C,
A, T, Q, N, S, G, D,
R, K, and H
P, A, S, Y, V, T, G, I,
D, N, R, G, Y, E, Q,
S, R, E, A, N, T, G, P,
G, S, R, K, A, N, Q,
H, E, D, P, W, L, and
F
T, Q, S, A, G, P, Y, I,
S, T, G, A, V, I, R, E,
C
S, N, R, T, G, K, E, H,
G, S, R, K, N, T, Q,
H, E, D, P, I, and L
T, S, G, D, C, A, L, N,
V, A, T, S, G, I, R, P,
A, T, G, S, Q, N, R,
Y, E, H, M, V, W, I,
L, and F
D, E, Q, N, S, T, K,
A, Y, P, M, W, I, F,
and L
A, T, C, G, S, N, P, R,
K, D, H, M, and F
L, F, I, V, P, A, T, Q,
S, G, R, K, H, M, Y,
E, D, N, T, R, K, G,
A, and V
A, G, Q, T, S, N, P, R,
Y, C, N, I, F, and L
S, T, G, H, A, P, Y, V,
I, V, and L
14-25
G, S, R, K, A, N, T,
Q, E, D, P, and Y
I, V, L, F, W, Y, A, T,
T, Q, N, G, C, M, S,
−1-34
S, N, Q, R, G, K, E,
A, C, G, S, Q, N, R,
Y, E, K, D, H, M, V,
I, and L
V, I, A, T, S, G, R, P,
S, N, Q, R, T, G, K, E,
K, R, H, S, G, N, Q,
K, R, H, S, G, N, Q,
D, E, Q, N, S, H, T, R,
A, T, C, G, S, Q, N, P,
In some embodiments, amino acid positions N1-N66 may be omitted or repeated up to 2 extra time (i.e., be included 0 to 3 times), each repeat being independently selected from the indicated amino acids. It is to be understood that the omission or repetition of any amino acid positions N1-N66 is independent of the omission or repetition of any amino acid at an alternate position. In some embodiments, amino acid positions N67-N71 may be repeated up to 1 extra time (i.e., be included 1 to 2 times), each repeat being independently selected from the indicated amino acids. It is to be understood that the repetition of any amino acid positions N67-N71 is independent of the repetition of any amino acid at an alternate position.
In some embodiments, each N1 is, independently, absent. In some embodiments, each N1 is, independently, an amino acid selected from the group consisting of S, N, D, Q, R, T, G, E, H, A, P, M, V, K, Y, W, F, L, I, and C. In some embodiments, each N1 is, independently, S. In some embodiments, each N2 is, independently, absent. In some embodiments, each N2 is, independently, an amino acid selected from the group consisting of P, A, S, Y, V, T, G, I, E, and C. In some embodiments, each N2 is, independently, P. In some embodiments, each N3 is, independently, absent. In some embodiments, each N3 is, independently, an amino acid selected from the group consisting of T, S, G, D, C, A, L, N, R, P, Y, V, W, I, and F. In some embodiments, each N3 is, independently, T. In some embodiments, each N4 is, independently, absent. In some embodiments, each N4 is, independently, an amino acid selected from the group consisting of S, R, E, A, Q, K, N, D, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N4 is, independently, S. In some embodiments, each N5 is, independently, absent. In some embodiments, each N5 is, independently, an amino acid selected from the group consisting of T, Q, N, G, C, M, S, A, E, D, Y, V, I, F, L, and W. In some embodiments, each N5 is, independently, T. In some embodiments, each N6 is, independently, absent. In some embodiments, each N6 is, independently, an amino acid selected from the group consisting of I, V, L, F, W, Y, A, T, S, E, D, and H. In some embodiments, each N6 is, independently, an amino acid selected from the group consisting of I and V. In some embodiments, each N7 is, independently, absent. In some embodiments, each N7 is, independently, an amino acid selected from the group consisting of P, V, A, S, N, G, E, L, and K. In some embodiments, each N8 is, independently, absent. In some embodiments, each N8 is, independently, an amino acid selected from the group consisting of A, G, Q, T, S, N, P, R, D, V, K, C, Y, W, I, L, and F. In some embodiments, each N8 is, independently, an amino acid selected from the group consisting of A, G, and Q. In some embodiments, each N9 is, independently, absent. In some embodiments, each N9 is, independently, an amino acid selected from the group consisting of F, Y, A, T, N, and R. In some embodiments, each N9 is, independently, an amino acid selected from the group consisting of F and Y. In some embodiments, each N10 is, independently, absent. In some embodiments, each N10 is, independently, an amino acid selected from the group consisting of T, Q, N, R, K, M, S, E, D, H, P, V, W, I, F, and L. In some embodiments, each N10 is, independently, T. In some embodiments, each N11 is, independently, absent. In some embodiments, each N11 is, independently, an amino acid selected from the group consisting of A, G, Q, T, S, N, P, R, D, V, K, C, Y, W, I, L, and F. In some embodiments, each N11 is, independently, an amino acid selected from the group consisting of A, G, and Q. In some embodiments, each N12 is, independently, absent. In some embodiments, each N12 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, G, K, E, H, D, A, P, L, M, V, Y, W, F, I, and C. In some embodiments, each N12 is, independently, S. In some embodiments, each N13 is, independently, absent. In some embodiments, each N13 is, independently, an amino acid selected from the group consisting of L, F, I, W, V, M, Y, C, A, T, Q, N, S, G, E, D, and R. In some embodiments, each N14 is, independently, absent. In some embodiments, each N14 is, independently, an amino acid selected from the group consisting of V, I, L, A, T, S, G, R, P, Y, N, H, C, M, F, Q, E, K, and D. In some embodiments, each N14 is, independently, V. In some embodiments, each N15 is, independently, absent. In some embodiments, each N15 is, independently, an amino acid selected from the group consisting of S, N, Q, T, G, K, E, H, D, A, C, P, Y, I, F, L, R, M, V, and W. In some embodiments, each N15 is, independently, S. In some embodiments, each N16 is, independently, absent. In some embodiments, each N16 is, independently, an amino acid selected from the group consisting of T, N, S, A, D, R, P, Y, V, W, I, F, and L. In some embodiments, each N16 is, independently, T. In some embodiments, each N17 is, independently, absent. In some embodiments, each N17 is, independently, an amino acid selected from the group consisting of S, N, Q, R, K, E, D, A, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N17 is, independently, S. In some embodiments, each N18 is, independently, absent. In some embodiments, each N18 is, independently, an amino acid selected from the group consisting of V, A, T, S, G, R, W, I, C, L, F, E, D, K, P, Y, N, H, M, and Q. In some embodiments, each N18 is, independently, V. In some embodiments, each N19 is, independently, absent. In some embodiments, each N19 is, independently, an amino acid selected from the group consisting of T, Q, N, S, A, E, G, D, Y, M, V, I, F, L, and W. In some embodiments, each N19 is, independently, T. In some embodiments, each N20 is, independently, absent. In some embodiments, each N20 is, independently, an amino acid selected from the group consisting of S, Q, R, K, E, A, N, D, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N20 is, independently, S. In some embodiments, each N21 is, independently, absent. In some embodiments, each N21 is, independently, an amino acid selected from the group consisting of V, W, I, C, L, F, A, T, S, E, D, K, G, R, P, Y, N, H, M, and Q. In some embodiments, each N21 is, independently, V. In some embodiments, each N22 is, independently, absent. In some embodiments, each N22 is, independently, an amino acid selected from the group consisting of T, Q, N, S, A, D, C, K, P, Y, M, V, W, I, F, G, E, H, R, and L. In some embodiments, each N22 is, independently, T. In some embodiments, each N23 is, independently, absent. In some embodiments, each N23 is, independently, an amino acid selected from the group consisting of L, F, I, V, P, A, T, Q, S, G, R, K, H, M, Y, and D. In some embodiments, each N23 is, independently, an amino acid selected from the group consisting of L, F, I, V, P, A, T, Q, S, G, R, K, and H. In some embodiments, each N24 is, independently, absent. In some embodiments, each N24 is, independently, an amino acid selected from the group consisting of T, Q, S, A, G, P, Y, I, K, H, V, F, L, N, D, C, M, W, E, and R. In some embodiments, each N24 is, independently, T. In some embodiments, each N25 is, independently, absent. In some embodiments, each N25 is, independently, an amino acid selected from the group consisting of S, R, E, A, Q, K, N, D, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N25 is, independently, S. In some embodiments, each N26 is, independently, absent. In some embodiments, each N26 is, independently, an amino acid selected from the group consisting of T, N, D, S, A, R, P, Y, V, W, I, F, and L. In some embodiments, each N26 is, independently, T. In some embodiments, each N27 is, independently, absent. In some embodiments, each N27 is, independently, an amino acid selected from the group consisting of D, N, R, E, Q, S, H, T, K, G, W, I, P, and Y. In some embodiments, each N27 is, independently, an amino acid selected from the group consisting of D and N. In some embodiments, each N28 is, independently, absent. In some embodiments, each N28 is, independently, an amino acid selected from the group consisting of V, A, T, S, G, R, W, I, C, L, F, E, D, K, P, Y, N, H, M, and Q. In some embodiments, each N28 is, independently, V. In some embodiments, each N29 is, independently, absent. In some embodiments, each N29 is, independently, an amino acid selected from the group consisting of T, S, A, D, C, L, N, R, P, Y, V, W, I, and F. In some embodiments, each N29 is, independently, T. In some embodiments, each N30 is, independently, absent. In some embodiments, each N30 is, independently, an amino acid selected from the group consisting of P, Y, V, A, T, S, G, I, E, and C. In some embodiments, each N30 is, independently, P. In some embodiments, each N31 is, independently, absent. In some embodiments, each N31 is, independently, an amino acid selected from the group consisting of T, Q, S, A, G, K, H, P, Y, V, I, F, L, N, D, C, M, W, E, and R. In some embodiments, each N31 is, independently, T. In some embodiments, each N32 is, independently, absent. In some embodiments, each N32 is, independently, an amino acid selected from the group consisting of S, R, E, A, Q, K, N, D, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N32 is, independently, S. In some embodiments, each N33 is, independently, absent. In some embodiments, each N33 is, independently, an amino acid selected from the group consisting of E, D, Q, N, S, T, H, R, G, A, P, F, and L. In some embodiments, each N34 is, independently, absent. In some embodiments, each N34 is, independently, an amino acid selected from the group consisting of D, N, R, E, Q, S, H, T, K, G, W, I, P, and Y. In some embodiments, each N34 is, independently, an amino acid selected form the group consisting of D and N. In some embodiments, each N35 is, independently, absent. In some embodiments, each N35 is, independently, an amino acid selected from the group consisting of T, Q, S, A, G, P, Y, I, K, H, V, F, L, N, D, C, M, W, E, and R. In some embodiments, each N35 is, independently, T. In some embodiments, each N36 is, independently, absent. In some embodiments, each N36 is, independently, an amino acid selected from the group consisting of G, S, K, A, T, Q, D, C, P, Y, V, W, I, L, and F. In some embodiments, each N37 is, independently, absent. In some embodiments, each N37 is, independently, an amino acid selected from the group consisting of F, Y, A, T, N, and R. In some embodiments, each N37 is, independently, an amino acid selected from the group consisting of F and Y. In some embodiments, each N38 is, independently, absent. In some embodiments, each N38 is, independently, an amino acid selected from the group consisting of V, A, T, S, G, R, W, I, C, L, F, E, D, K, P, Y, N, H, M and Q. In some embodiments, each N38 is, independently, V. In some embodiments, each N39 is, independently, absent. In some embodiments, each N39 is, independently, an amino acid selected from the group consisting of L, F, I, W, V, M, C, A, T, Q, N, S, G, D, R, K, and H. In some embodiments, each N40 is, independently, absent. In some embodiments, each N40 is, independently, an amino acid selected from the group consisting of P, A, S, Y, V, T, G, I, E, and C. In some embodiments, each N40 is, independently, P. In some embodiments, each N41 is, independently, absent. In some embodiments, each N41 is, independently, an amino acid selected from the group consisting of D, N, R, G, Y, E, Q, S, H, T, K, W, and I. In some embodiments, each N41 is, independently, an amino acid selected from the group consisting of D and N. In some embodiments, each N42 is, independently, absent. In some embodiments, each N42 is, independently, an amino acid selected from the group consisting of S, R, E, A, N, T, G, P, V, Q, K, H, D, Y, M, I, F, L, C, and W. In some embodiments, each N42 is, independently, S. In some embodiments, each N43 is, independently, absent. In some embodiments, each N43 is, independently, an amino acid selected from the group consisting of G, S, R, K, A, N, Q, H, E, D, P, W, L, and F. In some embodiments, each N44 is, independently, absent. In some embodiments, each N44 is, independently, an amino acid selected from the group consisting of T, Q, S, A, G, P, Y, I, N, E, D, C, K, H, R, V, L, M, F, and W. In some embodiments, each N44 is, independently, T. In some embodiments, each N45 is, independently, absent. In some embodiments, each N45 is, independently, an amino acid selected from the group consisting of S, T, G, A, V, I, R, E, N, P, Q, K, H, D, Y, M, F, L, C, and W. In some embodiments, each N45 is, independently, S. In some embodiments, each N46 is, independently, absent. In some embodiments, each N46 is, independently, C. In some embodiments, each N47 is, independently, absent. In some embodiments, each N47 is, independently, an amino acid selected from the group consisting of S, N, R, T, G, K, E, H, D, A, P, Y, V, W, I, L, Q, M, F, and C. In some embodiments, each N47 is, independently, S. In some embodiments, each N48 is, independently, absent. In some embodiments, each N48 is, independently, an amino acid selected from the group consisting of G, S, R, K, N, T, Q, H, E, D, P, I, and L. In some embodiments, each N49 is, independently, absent. In some embodiments, each N49 is, independently, an amino acid selected from the group consisting of T, S, G, D, C, A, L, N, R, P, Y, V, W, I, and F. In some embodiments, each N49 is, independently, T. In some embodiments, each N50 is, independently, absent. In some embodiments, each N50 is, independently, an amino acid selected from the group consisting of V, A, T, S, G, I, R, P, Y, L, N, H, C, M, F, Q, E, and K. In some embodiments, each N50 is, independently, V. In some embodiments, each N51 is, independently, absent. In some embodiments, each N51 is, independently, an amino acid selected from the group consisting of A, T, G, S, Q, N, R, Y, E, H, M, V, W, I, L, and F. In some embodiments, each N52 is, independently, absent. In some embodiments, each N52 is, independently, an amino acid selected from the group consisting of D, E, Q, N, S, T, K, A, Y, P, M, W, I, F, and L. In some embodiments, each N53 is, independently, absent. In some embodiments, each N53 is, independently, an amino acid selected from the group consisting of A, T, C, G, S, N, P, R, K, D, H, M, and F. In some embodiments, each N54 is, independently, absent. In some embodiments, each N54 is, independently, an amino acid selected from the group consisting of L, F, I, V, P, A, T, Q, S, G, R, K, H, M, Y, and D. In some embodiments, each N54 is, independently, an amino acid selected from the group consisting of L, F, I, V, P, A, T, Q, S, G, R, K, and H. In some embodiments, each N55 is, independently, absent. In some embodiments, each N55 is, independently, an amino acid selected from the group consisting of E, D, N, T, R, K, G, A, and V. In some embodiments, each N56 is, independently, absent. In some embodiments, each N56 is, independently, an amino acid selected from the group consisting of A, G, Q, T, S, N, P, R, D, V, W, K, C, Y, I, L, and F. In some embodiments, each N56 is, independently, an amino acid selected from the group consisting of A, G, and Q. In some embodiments, each N57 is, independently, absent. In some embodiments, each N57 is, independently, an amino acid selected from the group consisting of Y, C, N, I, F, and L. In some embodiments, each N58 is, independently, absent. In some embodiments, each N58 is, independently, an amino acid selected from the group consisting of S, T, G, H, A, P, Y, V, F, L, N, R, K, E, D, W, I, Q, M, and C. In some embodiments, each N58 is, independently, S. In some embodiments, each N59 is, independently, absent. In some embodiments, each N59 is, independently, an amino acid selected from the group consisting of I, V, and L. In some embodiments, each N59 is, independently, an amino acid selected from the group consisting of I and V. In some embodiments, each N60 is, independently, absent. In some embodiments, each N60 is, independently, S. In some embodiments, each N61 is, independently, absent. In some embodiments, each N61 is, independently, an amino acid selected from the group consisting of G, S, R, K, A, N, T, Q, E, D, P, and Y. In some embodiments, each N62 is, independently, absent. In some embodiments, each N62 is, independently, an amino acid selected from the group consisting of I, V, L, F, W, Y, A, T, S, E, D, and H. In some embodiments, each N62 is, independently, an amino acid selected from the group consisting of I and V. In some embodiments, each N63 is, independently, absent. In some embodiments, each N63 is, independently, an amino acid selected from the group consisting of T, Q, N, G, C, M, S, A, E, D, Y, V, I, F, L, and W. In some embodiments, each N63 is, independently, T. In some embodiments, each N64 is, independently, absent. In some embodiments, each N64 is, independently, an amino acid selected from the group consisting of S, N, Q, R, G, K, E, D, P, Y, W, F, T, H, A, V, L, I, M, and C. In some embodiments, each N64 is, independently, S. In some embodiments, each N65 is, independently, absent. In some embodiments, each N65 is, independently, an amino acid selected from the group consisting of A, C, G, S, Q, N, R, Y, E, K, D, H, M, V, I, and L. In some embodiments, each N66 is, independently, absent. In some embodiments, each N66 is, independently, an amino acid selected from the group consisting of V, I, A, T, S, G, R, P, Y, L, N, H, C, M, F, Q, E, K, and D. In some embodiments, each N66 is, independently, V. In some embodiments, each N67 is, independently, an amino acid selected from the group consisting of S, N, Q, R, T, G, K, E, H, D, A, C, P, Y, M, V, W, I, F, and L. In some embodiments, each N67 is, independently, an amino acid selected from the group consisting of S, N, Q, R, and T. In some embodiments, each N68 is, independently, an amino acid selected from the group consisting of K, R, H, S, G, N, Q, D, E, T, A, C, P, Y, M, V, W, I, L, and F. In some embodiments, each N68 is, independently, an amino acid selected from the group consisting of K, R, H, and S. In some embodiments, each N69 is, independently, an amino acid selected from the group consisting of K, R, H, S, G, N, Q, D, E, T, A, C, P, Y, M, V, W, I, L, and F. In some embodiments, each N69 is, independently, an amino acid selected from the group consisting of K, R, H, and S. In some embodiments, each N70 is, independently, an amino acid selected from the group consisting of D, E, Q, N, S, H, T, R, K, G, A, C, Y, P, M, V, W, I, F, and L. In some embodiments, each N70 is, independently, an amino acid selected from the group consisting of D, E, Q, and N. In some embodiments, each N71 is, independently, an amino acid selected from the group consisting of A, T, C, G, S, Q, N, P, R, Y, E, K, D, H, M, V, W, I, L, and F. In some embodiments, each N71 is, independently, an amino acid selected from the group consisting of A, T, C, and G.
In some embodiments, the pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO. 75.
In some embodiments, a synthetic pre-protein signal peptide is provided. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula I. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula II. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula III. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula IV. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula V. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula IX. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula XIII.
In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, the pre-protein signal peptide further comprises an amino acid sequence of SEQ ID NO. 68, SEQ ID NO. 69, or Formula XII.
In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 3. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 4. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 31. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 32. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 70. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 72. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 73
In some embodiments, a synthetic pro-protein signal peptide is provided. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence selected from the group consisting of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, and Formula XV. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula VI. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula VII. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula VIII. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula X. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula XI. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula XIV. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula XV.
In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 34, 35, 36, 37, 38, 56, 57, 58, 74, and 75. In some embodiments, the pro-protein signal peptide further comprises an amino acid sequence of SEQ ID NO. 68, SEQ ID NO. 69, or Formula XII.
In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 21. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 22. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 23. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 34. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 35. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 36. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 38. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 56. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 58. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 74. In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of SEQ ID NO: 75.
In some embodiments, a pre-protein plus a pro-protein signal peptide is provided. In some embodiments, the pre-protein plus a pro-protein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence of SEQ ID NO: 30.
In some embodiments, a recombinant polypeptide is provided, the recombinant polypeptide comprising a formula of (X1)n-(Y1)m-Z1, wherein X1 is a synthetic pre-protein signal peptide, Y1 is a synthetic pro-protein signal peptide, and Z1 is a payload protein, wherein n is 0 or 1, and m is 0 or 1, wherein n and m cannot concurrently be 0. In some embodiments, n is 0, m is 1, and the recombinant polypeptide comprises a formula of (Y1)-Z1. In some embodiments, n is 1, m is 0, and the recombinant polypeptide comprises a formula of (X1)-Z1. In some embodiments, n is 1, m is 1, and the recombinant polypeptide comprises a formula of (X1)-(Y1)-Z1.
In some embodiments, the recombinant polypeptide further comprises an amino acid sequence of SEQ ID NO. 68, SEQ ID NO. 69, or Formula XII at the N-terminus of the payload protein Z1. In some embodiments, the formula of (X1)n-(Y1)m-Z1 could further be written of (X1)n-(Y1)m-(K1)p-Z1, wherein X1 is a synthetic pre-protein signal peptide, Y1 is a synthetic pro-protein signal peptide, K1 is the a sequence selected from the group consisting of SEQ ID NO. 68, SEQ ID NO. 69, and Formula XII, and Z1 is a payload protein, wherein n is 0 or 1, m is 0 or 1, and p is 0 or 1, and wherein n and m cannot concurrently be 0. In some embodiments, n is 0, m is 1, p is 0 and the recombinant polypeptide comprises a formula of (Y1)-Z1. In some embodiments, n is 0, m is 1, p is 1 and the recombinant polypeptide comprises a formula of (Y1)-(K1)-Z1. In some embodiments, n is 1, m is 0, p is 0 and the recombinant polypeptide comprises a formula of (X1)-Z1. In some embodiments, n is 1, m is 0, p is 1 and the recombinant polypeptide comprises a formula of (X1)-(K1)-Z1. In some embodiments, n is 1, m is 1, p is 0 and the recombinant polypeptide comprises a formula of (X1)-(Y1)-Z1. In some embodiments, n is 1, m is 1, p is 1 and the recombinant polypeptide comprises a formula of (X1)-(Y1)-(K1)-Z1.
In some embodiments, n is 1 and X1 comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII. In some embodiments, X1 comprises an amino acid sequence of Formula I. In some embodiments, X1 comprises an amino acid sequence of Formula II. In some embodiments, X1 comprises an amino acid sequence of Formula III. In some embodiments, X1 comprises an amino acid sequence of Formula IV. In some embodiments, X1 comprises an amino acid sequence of Formula V. In some embodiments, X1 comprises an amino acid sequence of Formula IX. In some embodiments, X1 comprises an amino acid sequence of Formula XIII. In some embodiments, X1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, X1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, X1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73.
In some embodiments, m is 1 and Y1 comprises an amino acid sequence selected from the group consisting of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, and Formula XV. In some embodiments, Y1 comprises an amino acid sequence of Formula VI. In some embodiments, Y1 comprises an amino acid sequence of Formula VII. In some embodiments, Y1 comprises an amino acid sequence of Formula VIII. In some embodiments, Y1 comprises an amino acid sequence of Formula X. In some embodiments, Y1 comprises an amino acid sequence of Formula XI. In some embodiments, Y1 comprises an amino acid sequence of Formula XIV. In some embodiments, Y1 comprises an amino acid sequence of Formula XV. In some embodiments, Y1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, Y1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, Y1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75.
In some embodiments, X1 and Y1 are combined and represented by pre-protein plus a pro-protein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence of SEQ ID NO: 30.
In some embodiments, the Z1 is any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an antiviral, insulin, an incretin, an enzyme, an enzyme inhibitor, a hormone, a cytokine, an antibody, an antimicrobial peptide, a mucosal protein, pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 59:
or is substantially similar to SEQ ID NO. 59 or is an active fragment of SEQ ID NO. 59. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 59. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 59.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 60:
or is substantially similar to SEQ ID NO. 60 or is an active fragment of SEQ ID NO. 60. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 60. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 60.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 61:
or is substantially similar to SEQ ID NO. 61 or is an active fragment of SEQ ID NO. 61. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 61. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 61.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 62:
or is substantially similar to SEQ ID NO. 62 or is an active fragment of SEQ ID NO. 62. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 62. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 62.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 63:
or is substantially similar to SEQ ID NO. 63 or is an active fragment of SEQ ID NO. 63. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 63. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 63.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 64:
or is substantially similar to SEQ ID NO. 64 or is an active fragment of SEQ ID NO. 64. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 64. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 64.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 65:
or is substantially similar to SEQ ID NO. 65 or is an active fragment of SEQ ID NO. 65. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 65. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 65.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 66:
or is substantially similar to SEQ ID NO. 66 or is an active fragment of SEQ ID NO. 66. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 66. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 66.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 67:
or is substantially similar to SEQ ID NO. 67 or is an active fragment of SEQ ID NO. 67. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 67. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 67.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO. 85:
or is substantially similar to SEQ ID NO. 85 or is an active fragment of SEQ ID NO. 85. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 85. In some embodiments, Z1 comprises an amino acid sequence of SEQ ID NO. 85.
In any of the embodiments herein, Z1 may further comprise an affinity tag. The affinity tag may be utilized, for example, for protein purification or detection. The affinity tag may be utilized for any method known in the art for which affinity tags are utilized. Affinity tags are known in the art, and any such affinity tag may be utilized. Non-limiting examples of affinity tags that may be utilized include 6×HIS (SEQ ID NO: 105), FLAG, GST, MBP, a streptavidin peptide, GFP, and the like. In some embodiments, any peptide sequence that can be utilized for purification or detection may be utilized.
In some embodiments, the recombinant polypeptide comprises a formula of (X1)n-(Y1)m-Z1, wherein n is 0 or 1 and m is 0 or 1, wherein n and m cannot concurrently be 0, wherein X1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73, Y1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75, and Z1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, 66, 67, and 85. In some embodiments, the components X1, Y1, and Z1 are fused directly. In some embodiments, the components X1, Y1, and Z1, are fused indirectly via, for example, a peptide linker as provided for herein.
In some embodiments, the recombinant polypeptide further comprises an amino acid sequence of SEQ ID NO. 68 at the N-terminus of the payload protein Z1. In some embodiments, the formula of (X1)n-(Y1)m-Z1 could further be written of (X1)n-(Y1)m-(K1)p-Z1, wherein X1 is a synthetic pre-protein signal peptide, Y1 is a synthetic pro-protein signal peptide, K1 is a sequence selected from the group consisting of SEQ ID NO. 68, SEQ ID NO. 69, and Formula XII, and Z1 is a payload protein, wherein n is 0 or 1, m is 0 or 1, and p is 0 or 1, and wherein n and m cannot concurrently be 0. In some embodiments, n is 0, m is 1, p is 0 and the recombinant polypeptide comprises a formula of (Y1)-Z1. In some embodiments, n is 0, m is 1, p is 1 and the recombinant polypeptide comprises a formula of (Y1)-(K1)-Z1. In some embodiments, n is 1, m is 0, p is 0 and the recombinant polypeptide comprises a formula of (X1)-Z1. In some embodiments, n is 1, m is 0, p is 1 and the recombinant polypeptide comprises a formula of (X1)-(K1)-Z1. In some embodiments, n is 1, m is 1, p is 0 and the recombinant polypeptide comprises a formula of (X1)-(Y1)-Z1. In some embodiments, n is 1, m is 1, p is 1 and the recombinant polypeptide comprises a formula of (X1)-(Y1)-(K1)-Z1.
In some embodiments, a nucleic acid is provided. In some embodiments, the nucleic acid encodes for a recombinant polypeptide as provided for herein. In some embodiments, the recombinant polypeptide comprises a synthetic signal peptide and a payload protein. In some embodiments, the synthetic signal peptide is as provided for herein. In some embodiments, the payload protein is as provided for herein.
In some embodiments, an engineered yeast is provided. In some embodiments, the engineered yeast is genetically modified with a nucleic acid encoding a recombinant polypeptide having a formula of (X1)n-(Y1)m-Z1, wherein X1 is a synthetic pre-protein signal peptide, Y1 is a synthetic pro-protein signal peptide, Z1 is a payload protein, n is 0 or 1, m is 0 or 1, and n and m cannot concurrently be 0.
In some embodiments, the recombinant polypeptide further comprises an amino acid sequence of SEQ ID NO. 68 at the N-terminus of the payload protein Z1. In some embodiments, the formula of (X1)n-(Y1)m-Z1 could further be written of (X1)n-(Y1)m-(K1)p-Z1, wherein X1 is a synthetic pre-protein signal peptide, Y1 is a synthetic pro-protein signal peptide, K1 is a sequence selected from the group consisting of SEQ ID NO. 68, SEQ ID NO. 69, and Formula XII, and Z1 is a payload protein, wherein n is 0 or 1, m is 0 or 1, and p is 0 or 1, and wherein n and m cannot concurrently be 0. In some embodiments, n is 0, m is 1, p is 0 and the recombinant polypeptide comprises a formula of (Y1)-Z1. In some embodiments, n is 0, m is 1, p is 1 and the recombinant polypeptide comprises a formula of (Y1)-(K1)-Z1. In some embodiments, n is 1, m is 0, p is 0 and the recombinant polypeptide comprises a formula of (X1)-Z1. In some embodiments, n is 1, m is 0, p is 1 and the recombinant polypeptide comprises a formula of (X1)-(K1)-Z1. In some embodiments, n is 1, m is 1, p is 0 and the recombinant polypeptide comprises a formula of (X1)-(Y1)-Z1. In some embodiments, n is 1, m is 1, p is 1 and the recombinant polypeptide comprises a formula of (X1)-(Y1)-(K1)-Z1
In some embodiments, n is 1 and X1 comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII. In some embodiments, X1 comprises an amino acid sequence of Formula I. In some embodiments, X1 comprises an amino acid sequence of Formula II. In some embodiments, X1 comprises an amino acid sequence of Formula III. In some embodiments, X1 comprises an amino acid sequence of Formula IV. In some embodiments, X1 comprises an amino acid sequence of Formula V. In some embodiments, X1 comprises an amino acid sequence of Formula IX. In some embodiments, X1 comprises an amino acid sequence of Formula XIII. In some embodiments, X1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, X1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, X1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73.
In some embodiments, m is 1 and Y1 comprises an amino acid sequence selected from the group consisting of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, and Formula XV. In some embodiments, Y1 comprises an amino acid sequence of Formula VI. In some embodiments, Y1 comprises an amino acid sequence of Formula VII. In some embodiments, Y1 comprises an amino acid sequence of Formula VIII. In some embodiments, Y1 comprises an amino acid sequence of Formula X. In some embodiments, Y1 comprises an amino acid sequence of Formula XI. In some embodiments, Y1 comprises an amino acid sequence of Formula XIV. In some embodiments, Y1 comprises an amino acid sequence of Formula XV. In some embodiments, Y1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, Y1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, Y1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75.
In some embodiments, the Z1 is any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an antiviral, insulin, an incretin, an enzyme, an enzyme inhibitor, a hormone, a cytokine, an antibody, an antimicrobial peptide, a mucosal protein, pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein.
In some embodiments, Z1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, 66, and 67. In some embodiments, Z1 comprises an amino acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, 66, 67, and 85. In some embodiments, Z1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, 66, 67, and 85.
In some embodiments, the components X1, Y1, and Z1 are fused directly. In some embodiments, the components X1, Y1, and Z1, are fused indirectly via, for example, a peptide linker as provided for herein.
In some embodiments, the identity of X1, Y1, and Z1 are influenced by the strain of yeast utilized. In some embodiments, the strain of yeast is any yeast as provided for herein. In some embodiments, the yeast is selected from the group consisting of Kluyveromyces, Pichia, Saccharomyces, Trichoderma, and Aspergillus. Specific yeast, X1, Y1, and Z1 combinations are described and provided for below. It is to be understood that the embodiments provided below are merely exemplary and are not meant to limit the scope of the invention in any way. Thus, although a particular embodiment may be silent on the use of a particular pre or pro protein SEQ ID NO, this is not to be construed as the particular SEQ ID NO. being excluded from use in the particular yeast. Further, although a particular embodiment may be silent on the inclusion of any synthetic pre or pro protein signal peptides, this is not to be construed as the pre or pro protein signal peptides are excluded from use in the particular yeast. For example, if a recombinant polypeptide is described for use in a particular yeast and the recombinant polypeptide is said to comprise a synthetic pre-protein signal peptide domain and a payload protein domain, this is not to be construed as a synthetic pro-protein signal domain cannot be included for the particular yeast. Likewise, if a recombinant polypeptide is described for use is a particular yeast and the recombinant polypeptide is said to comprise a synthetic pro-protein signal peptide domain and a payload protein domain, this is not to be construed as a synthetic pre-protein signal domain cannot be included for the particular yeast.
Synthetic Pre-Protein Signal Peptides and their Use in Kluyveromyces Yeast
In some embodiments, a synthetic pre-protein signal peptide that may be fused to a payload protein to facilitate secretion of the payload protein from Kluyveromyces yeast (e.g., K. lactis) is provided. In some embodiments, Kluyveromyces yeast (e.g., K. lactis) may be genetically modified with a nucleic acid molecule encoding for expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused either directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula I or SEQ ID NO. 1. In some embodiments, the nucleic acid molecule is any nucleic acid molecule encoding for a peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1. For example, SEQ ID NO. 39 may be used to encode for the synthetic pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO. 1. It is to be understood that the previous example is not meant to be limiting in any way. One who is skilled in the art will understand how to develop a suitable nucleotide sequence that will induce expression of a synthetic signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1. In some embodiments, a signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 may be fused directly or indirectly to a native constitutive pro-protein signal peptide or a synthetic signal peptide as disclosed herein.
In some embodiments, a recombinant polypeptide comprising a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and a payload protein is provided. In some embodiments, inclusion of the pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in another embodiment, a method of producing a payload protein with Kluyveromyces yeast (e.g., K. lactis) is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic signal peptide comprising an amino acid sequence of Formula I SEQ ID NO. 1; genetically modifying the Kluyveromyces yeast (e.g., K. lactis) with the nucleic acid molecule, thereby generating engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding the synthetic signal peptide of SEQ ID NO. 1 is SEQ ID NO. 39. In some embodiments, the nucleic acid molecule encoding the synthetic signal peptide amino acid of Formula I or SEQ ID NO. 1 is any nucleic acid molecule encoding for said amino acid sequences.
In some embodiments, a method of increasing extracellular secretion of a payload protein from Kluyveromyces yeast (e.g., K. lactis) is provided, the method comprising providing a nucleic acid encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide, genetically modifying the Kluyveromyces yeast (e.g., K. lactis) with the nucleic acid, thereby generating an engineered yeast, and culturing the engineered yeast under effective conditions to produce and secrete an increased amount of payload protein when compared to the amount of payload protein secreted by Kluyveromyces yeast (e.g., K. lactis) using a recombinant polypeptide comprising the payload protein and signal peptide α-MF or any other commonly utilized signal peptide such as SUC2, PHO5, or HSA. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula I or SEQ ID NO. 1. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is connected to the payload protein via a peptide linker as provided for herein.
In some embodiments, an engineered Kluyveromyces yeast (e.g., K. lactis) is provided, wherein the yeast is genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula I or SEQ ID NO. 1. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is indirectly fused to the payload protein via a connecting linker peptide sequence as provided for herein. In some embodiments, the nucleic acid molecule used to encode the synthetic pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO. 1 is given by SEQ ID NO. 39. In some embodiments, the nucleic acid molecule used to encode the synthetic pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 is any nucleic acid molecule encoding for said amino acid sequence.
In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine, an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to IGF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art.
Synthetic Pre-Protein Signal Peptides and their Use in a Pichia Yeast
In some embodiments, a synthetic pre-protein signal peptide for use in the yeast species Pichia (e.g., P. pastoris) is provided. In some embodiments, the Pichia yeast may be genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal comprises an amino acid sequence represented by Formula II or SEQ ID NOs. 2, 3, 4, 5, 6, or 7. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein, connecting via a peptide linker as provided for herein. In some embodiments, any nucleic acid encoding for Formula II or SEQ ID NO. 2, 3, 4, 5, 6 or 7 may be utilized to induce expression of the synthetic signal peptide. One of skill in the art will understand how to develop a suitable nucleotide sequence that will induce expression of a synthetic pre-protein signal represented by Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7. In some embodiments, the synthetic pre-protein signal peptide of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 may further be fused directly or indirectly to a native constitutive pro-protein signal peptide or a synthetic signal peptide as disclosed herein. In some embodiments, the synthetic pre-protein signal peptide is further fused to a native constitutive pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide is further fused to a synthetic signal peptide as disclosed herein. In some embodiments, the synthetic pre-protein signal peptide of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 is further fused to a synthetic pro-protein signal peptide selected from the group consisting of SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 34, 35, 36, 37, 38, 56, 57, and 58. In some embodiments, the synthetic pre-protein signal peptide of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 is further fused to a synthetic pro-protein signal peptide as represented by SEQ. ID NO. 17.
In some embodiments, a recombinant polypeptide comprising a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and a payload protein is provided. In some embodiments, inclusion of the pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in some embodiments, a method of producing a payload protein with Pichia yeast (e.g., P. pastoris) is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7; genetically modifying the a Pichia yeast (e.g., P. pastoris) with the nucleic acid molecule, thereby generating engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding for the amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 is any nucleic acid molecule encoding for said amino acid sequence.
In some embodiments, a method of increasing extracellular secretion of a payload protein from a Pichia yeast is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide; genetically modifying the a Pichia yeast (e.g., P. pastoris) with the nucleic acid, thereby generating an engineered yeast, and culturing the engineered yeast under effective conditions to produce and secrete an increased amount of payload protein when compared to the amount of payload protein secreted by a Pichia yeast genetically modified to express a recombinant polypeptide comprising the payload protein and pre-protein signal peptide α-MF (α-MF comprising an amino acid sequence represented by SEQ ID NO. 27). In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, an engineered Pichia yeast (e.g., P. pastoris) is provided, wherein the yeast is genetically modified with a nucleic acid encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein. In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine, an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to IGF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art.
Synthetic Pre-Protein Signal Peptides and their Use in Saccharomyces Yeast
In another embodiment, a synthetic pre-protein signal peptide for use in the yeast species Saccharomyces (e.g., S. boulardii or S. cerevisiae) is provided. In some embodiments, S. cerevisiae yeast may be genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein. In some embodiments, any nucleic acid encoding for Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 may be utilized to induce expression of the synthetic pre-protein signal peptide. One of skill in the art will understand how to develop a suitable nucleic acid that will induce expression of a synthetic signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 may be fused directly or indirectly to a native constitutive pro-protein signal peptide. In some embodiments, a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 may be fused directly or indirectly to a synthetic signal peptide as disclosed herein, such as Formula VI, Formula VII, Formula VIII or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the native or synthetic pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the native or synthetic pro-protein signal peptide via, for example, a peptide linker as provided for herein.
In some embodiments, a recombinant polypeptide comprising a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and a payload protein is provided. In some embodiments, inclusion of the synthetic pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in another embodiment, a method of producing a payload protein with Saccharomyces yeast is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16; genetically modifying the Saccharomyces yeast with the nucleic acid, thereby generating engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding for the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 is any nucleic acid molecule encoding for said amino acid sequence.
In some embodiments, a method of increasing extracellular secretion of a payload protein from Saccharomyces yeast is provided, the method comprising providing a nucleic acid encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide; genetically modifying the Saccharomyces yeast with the nucleic acid, thereby generating an engineered yeast, and culturing the engineered yeast under effective conditions to produce and secrete an increased amount of payload protein when compared to the amount of payload protein secreted by Saccharomyces yeast genetically modified to express a recombinant polypeptide comprising the payload protein and pre-protein signal peptide α-MF or Yeast Aspartic Protease 3 (YAP). In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, an engineered Saccharomyces yeast (e.g., S. boulardii or S. cerevisiae) is provided, wherein the yeast is genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein. In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine, an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to IGF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art.
Synthetic Pre-Protein Signal Peptides and their Use in Trichoderma Yeast
In some embodiments, a synthetic pre-protein signal peptide for use in the yeast species Trichoderma (e.g., T. reesei or T. viride) is provided. In some embodiments, Trichoderma yeast may be genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein. In some embodiments, any nucleic acid molecule encoding for Formula IX or SEQ ID NO. 31, 32, or 33 may be utilized to induce expression of the synthetic signal peptide. One of skill in the art will understand how to develop a suitable nucleotide sequence that will induce expression of a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33. In some embodiments, a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 may further be fused directly or indirectly to a native constitutive pro-protein signal peptide or a synthetic signal peptide as disclosed herein. In some embodiments, the synthetic pre-protein signal peptide is further fused to a native constitutive pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide is further fused to a synthetic signal peptide as disclosed herein.
In some embodiments, a recombinant polypeptide comprising a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and a payload protein is provided. In some embodiments, inclusion of the pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in another embodiment, a method of producing a payload protein with Trichoderma yeast (e.g., T. reesei or T. viride) is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33; genetically modifying the T. reesei yeast with the nucleic acid molecule, thereby generating engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding for the amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 is any nucleic acid molecule encoding for said amino acid sequence.
In some embodiments, a method of increasing extracellular secretion of a payload protein from a Trichoderma yeast (e.g., T. reesei or T. viride) is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide; genetically modifying the Trichoderma yeast with the nucleic acid, thereby generating an engineered yeast, and culturing the engineered yeast under effective conditions to secrete an increased amount of payload protein when compared to the amount of payload protein secreted by Trichoderma yeast genetically modified to express a recombinant polypeptide comprising the payload protein and pre-protein signal peptide comprising a native pre-protein signal peptide sequence as provided for herein or a control pre-protein signal peptide sequence as provided for herein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, an engineered Trichoderma yeast (e.g., T. reesei or T. viride) is provided, wherein the yeast is genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine, an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to IGF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art.
Synthetic Pre-Protein Signal Peptides and their Used is Aspergillus Yeast Strains
In some embodiments, a synthetic pre-protein signal peptide for use in the yeast species Aspergillus (e.g., A. niger) is provided. In some embodiments, Aspergillus yeast may be genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein. In some embodiments, any nucleic acid molecule encoding for Formula XIII or SEQ ID NO. 70, 71, 72, or 73 may be utilized to induce expression of the synthetic signal peptide. One of skill in the art will understand how to develop a suitable nucleotide sequence that will induce expression of a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73. In some embodiments, a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 may further be fused directly or indirectly to a native constitutive pro-protein signal peptide or a synthetic signal peptide as disclosed herein. In some embodiments, the synthetic pre-protein signal peptide is further fused to a native constitutive pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide is further fused to a synthetic signal peptide as disclosed herein.
In some embodiments, a recombinant polypeptide comprising a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and a payload protein is provided. In some embodiments, inclusion of the pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in another embodiment, a method of producing a payload protein with Aspergillus yeast (e.g., A. niger) is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73; genetically modifying the Aspergillus yeast with the nucleic acid molecule, thereby generating engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding for the amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 is any nucleic acid molecule encoding for said amino acid sequence.
In some embodiments, a method of increasing extracellular secretion of a payload protein from a Aspergillus yeast (e.g., A. niger) is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide; genetically modifying the Aspergillus yeast with the nucleic acid, thereby generating an engineered yeast, and culturing the engineered yeast under effective conditions to secrete an increased amount of payload protein when compared to the amount of payload protein secreted by Aspergillus yeast genetically modified to express a recombinant polypeptide comprising the payload protein and pre-protein signal peptide comprising a native pre-protein signal peptide sequence as provided for herein or a control pre-protein signal peptide. In some embodiments, the control pre-protein signal peptide is:
In some embodiments, the control pre-protein signal peptide is glucoamylaseprotein, as represented by SEQ ID NO. 77 below:
In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, an engineered Aspergillus yeast (e.g., A. niger) is provided, wherein the yeast is genetically modified with a nucleic acid molecule encoding the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide as provided for herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine, an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to IGF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art.
In some embodiments, various synthetic pro-protein signal peptides are provided that, in addition to suitability for use in combination with a pre-protein signal peptide as described above, may also be used without a synthetic pre-protein signal peptide. In some embodiments, a pro-protein signal peptide may comprise an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24, any of which may be used in any yeast strain as provided for herein, such as Saccharomyces (e.g., S. cerevisiae, S. boulardii), Pichia (e.g., P. pastoris), and/or Kluyveromyces (e.g., K. lactis). In some embodiments, a synthetic signal peptide may comprise only a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, a synthetic signal peptide may further comprise any native constitutive pre-protein signal peptide. In some embodiments, a synthetic signal peptide may further comprise any synthetic pre-protein signal peptides as described herein. In some embodiments, when used in combination with a pre-protein signal peptide (native or synthetic), the N-terminus of the pro-protein signal peptide may be fused directly or indirectly to the C-terminus of the pre-protein signal peptide. The pro-protein signal peptide may, in turn, may be fused directly or indirectly to the N-terminus of a payload protein, optionally through a KR site, Ste13 cleavage site, and/or spacer. In some embodiments, indirect fusion may be accomplished through, for example, inclusion of a linker peptide as provided for herein.
Accordingly, in some embodiments, a synthetic signal peptide is provided, the peptide comprising a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24 fused directly or indirectly to a payload protein. In some embodiments, the synthetic signal peptide further comprises a pre-protein signal peptide. In some embodiments, the pre-protein signal peptide is a native signal peptide. In some embodiments, the pre-protein signal peptide is a synthetic signal peptide. In some embodiments, the pre-protein signal peptide comprises an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16.
In some embodiments, a recombinant polypeptide comprising a synthetic pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24 and a payload protein is provided. In some embodiments, inclusion of the pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in another embodiment, a method of producing a payload protein with a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24; genetically modifying the yeast with the nucleic acid, thereby generating engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the yeast strain is selected from the group comprising Saccharomyces (e.g., S. cerevisiae, S. boulardii), Pichia (e.g., P. pastoris), and/or Kluyveromyces (e.g., K. lactis). In some embodiments, the nucleic acid molecule encoding for the amino acid sequence of Formula VI, Formula VII, Formula VIII or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24 is any nucleic acid molecule encoding for said amino acid sequence.
In some embodiments, a method of increasing extracellular secretion of a payload protein from a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pro-protein signal peptide; genetically modifying the yeast with the nucleic acid molecule, thereby generating an engineered yeast, and culturing the engineered yeast under effective conditions to secrete an increased amount of payload protein when compared to the amount of payload protein secreted by the yeast genetically modified to express a recombinant polypeptide comprising the payload protein and a native pro-protein signal peptide. In some embodiments, the yeast strain is selected from the group comprising Saccharomyces (e.g., S. cerevisiae, S. boulardii), Pichia (e.g., P. pastoris), and/or Kluyveromyces (e.g., K. lactis). In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the synthetic pro-protein further comprises a native pre-protein signal peptide. In some embodiments, the synthetic pro-protein further comprises a synthetic pre-protein signal peptide as provided for herein. In some embodiments, the synthetic pro-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pro-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine, an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to IGF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art.
In some embodiments, various synthetic pro-protein signal peptides are provided that, in addition to suitability for use in combination with a pre-protein signal peptide as described above, may also be used without a synthetic pre-protein signal peptide. In some embodiments, a pro-protein signal peptide may comprise an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, any of which may be used in any yeast species within the Trichoderma strain (e.g., T. reesei, T. viride). In some embodiments, a synthetic signal peptide may comprise only an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38. In some embodiments, the synthetic signal peptide may further comprise any native constitutive pre-protein signal peptide. In some embodiments, the synthetic signal peptide may further comprise any of the synthetic pre-protein signal peptides as provided for herein. In some embodiments, when used in combination with a pre-protein signal peptide (native or synthetic), the N-terminus of the pro-protein signal peptide may be fused directly or indirectly to the C-terminus of the pre-protein signal peptide. The pro-protein signal peptide may, in turn, may be fused directly or indirectly to the N-terminus of a payload protein, optionally through a KR site, Ste13 cleavage site, and/or spacer. In some embodiments, indirect fusion may be accomplished through, for example, inclusion of a linker peptide as provided for herein.
Accordingly, in some embodiments, a synthetic signal peptide is provided, the synthetic signal peptide comprising a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38 fused directly or indirectly to a payload protein. In some embodiments, the synthetic signal peptide further comprises a pre-protein signal peptide. In some embodiments, the pre-protein signal peptide is a native pre-protein signal peptide. In some embodiments, the pre-protein signal peptide is a synthetic pre-protein signal peptide as provided for herein. In some embodiments, the pre-protein signal peptide comprises an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33.
In some embodiments, a recombinant polypeptide comprising a synthetic pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38 and a payload protein is provided. In some embodiments, inclusion of the pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in another embodiment, a method of producing a payload protein with a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38; genetically modifying the yeast with the nucleic acid, thereby generating engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the yeast strain is a Trichoderma yeast strain (e.g., T. reesei, T. viride). In some embodiments, the nucleic acid molecule encoding for the amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38 is any nucleic acid molecule encoding for said amino acid sequence.
In some embodiments, a method of increasing extracellular secretion of a payload protein from a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pro-protein signal peptide; genetically modifying the yeast with the nucleic acid molecule, thereby generating an engineered yeast, and culturing the engineered yeast under effective conditions to secrete an increased amount of payload protein when compared to the amount of payload protein secreted by the yeast genetically modified to express a recombinant polypeptide comprising the payload protein and a native pro-protein signal peptide. In some embodiments, the yeast strain is a Trichoderma yeast strain (e.g., T. reesei, T. viride). In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38. In some embodiments, the synthetic pro-protein signal peptide further comprises a native pre-protein signal peptide. In some embodiments, the synthetic pro-protein signal peptide further comprises a synthetic pre-protein signal peptide as provided for herein. In some embodiments, the synthetic pro-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pro-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine, an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to IGF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art.
Synthetic Pro-Protein Signal Peptides and their Use in Aspergillus Yeast Strains
In some embodiments, various synthetic pro-protein signal peptides are provided that, in addition to suitability for use in combination with a pre-protein signal peptide as described above, may also be used without a synthetic pre-protein signal peptide. In some embodiments, a pro-protein signal peptide may comprise an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, any of which may be used in any yeast species within the Aspergillus strain (e.g., A. niger). In some embodiments, a synthetic signal peptide may comprise only an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75. In some embodiments, the synthetic signal peptide may further comprise any native constitutive pre-protein signal peptide. In some embodiments, the synthetic signal peptide may further comprise any of the synthetic pre-protein signal peptides as provided for herein. In some embodiments, when used in combination with a pre-protein signal peptide (native or synthetic), the N-terminus of the pro-protein signal peptide may be fused directly or indirectly to the C-terminus of the pre-protein signal peptide. The pro-protein signal peptide may, in turn, may be fused directly or indirectly to the N-terminus of a payload protein, optionally through a KR site, Ste13 cleavage site, and/or spacer. In some embodiments, indirect fusion may be accomplished through, for example, inclusion of a linker peptide as provided for herein.
Accordingly, in some embodiments, a synthetic signal peptide is provided, the synthetic signal peptide comprising a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75 fused directly or indirectly to a payload protein. In some embodiments, the synthetic signal peptide further comprises a pre-protein signal peptide. In some embodiments, the pre-protein signal peptide is a native pre-protein signal peptide. In some embodiments, the pre-protein signal peptide is a synthetic pre-protein signal peptide as provided for herein. In some embodiments, the pre-protein signal peptide comprises an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73.
In some embodiments, a recombinant polypeptide comprising a synthetic pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75 and a payload protein is provided. In some embodiments, inclusion of the pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75 will result in the payload protein being more readily secreted by the yeast in which it is produced. Accordingly, in another embodiment, a method of producing a payload protein with a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75; genetically modifying the yeast with the nucleic acid, thereby generating engineered yeast; and culturing the engineered yeast under effective conditions to express the recombinant polypeptide. In some embodiments, the yeast strain is an Aspergillus yeast strain (e.g., A. niger). In some embodiments, the nucleic acid molecule encoding for the amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75 is any nucleic acid molecule encoding for said amino acid sequence.
In some embodiments, a method of increasing extracellular secretion of a payload protein from a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pro-protein signal peptide; genetically modifying the yeast with the nucleic acid molecule, thereby generating an engineered yeast, and culturing the engineered yeast under effective conditions to secrete an increased amount of payload protein when compared to the amount of payload protein secreted by the yeast genetically modified to express a recombinant polypeptide comprising the payload protein and a native pro-protein signal peptide. In some embodiments, the yeast strain is a Aspergillus yeast strain (e.g., A. niger). In some embodiments, the synthetic pro-protein signal peptide comprises an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75. In some embodiments, the synthetic pro-protein signal peptide further comprises a native pre-protein signal peptide. In some embodiments, the synthetic pro-protein signal peptide further comprises a synthetic pre-protein signal peptide as provided for herein. In some embodiments, the synthetic pro-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pro-protein signal peptide is fused indirectly to the payload protein via, for example, a peptide linker as provided for herein.
In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an enzyme (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), a growth factor (e.g., IGF-1), insulin, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), a cytokine, an antibody, an antimicrobial peptide), a mucosal protein (e.g., trefoil factor, Reg3 protein, superoxide dismutase), an agricultural product (e.g., pesticide, bactericide herbicide, fungicide, nematicide, miticide, plant growth regulator, plant growth stimulator, or fertilizer), a vaccine, a diagnostic protein, a feed conversion enzyme, a flavoring, or a nutritional protein. The examples listed are provided for clarity only and are not meant to be limiting in any way. Thus, for example, the current disclosure is not limited to 8GF-1 for “growth factor”, but rather encompasses and includes all growth factors known in the art.
Provided herein are synthetic signal peptides that may be used to genetically modify a particular strain of yeast to increase secretion of any payload protein or peptide in that yeast. Various suitable signal peptides are disclosed above with specific examples of signal peptides comprising various synthetic pre- and synthetic pro-protein signal detailed in Table 17 below.
Kluyveromyces
Pichia,
Saccharomyces
Pichia
Pichia
Pichia
Pichia
Pichia
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Trichoderma
Trichoderma
Trichoderma
Pichia
Saccharomyces
Saccharomyces
Kluyveromyces
Pichia
Saccharomyces
Kluyveromyces
Pichia
Saccharomyces
Saccharomyces
Saccharomyces
Saccharomyces
Pichia,
Saccharomyces
Saccharomyces
Trichoderma
Trichoderma
Trichoderma
Trichoderma
Trichoderma
Saccharomyces
Kluyveromyces
Pichia
Kluyveromyces
Aspergillus
The suitable strains recited in the prior table are meant to be exemplary, not exclusionary Thus, the table should not be interpreted as suggesting that the “suitable strains” are the only strains for which the recited pre and pro protein signal peptides can be used. Rather, the “suitable strain” is merely an example of a strain in which the recited pre and pro protein signal peptides can be used.
In some embodiments, any synthetic signal sequence may comprise solely a synthetic pre-protein signal peptide (e.g., SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73) with no additional pro-protein signal peptide sequence. In some embodiments, any synthetic signal sequence may comprise a pre-protein signal peptide (e.g., SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73) fused to any native pro-protein peptide or portion thereof (e.g., pro-α-MF). In some embodiments, any synthetic signal sequence may comprise a pre-protein signal peptide (e.g., SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73) fused to any synthetic pro-protein signal peptide (e.g., SEQ ID NOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75) or portion thereof. In some embodiments, any synthetic signal sequence may comprise solely a synthetic pro-protein signal peptide (e.g., SEQ ID NOs 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75) with no additional pre-protein signal peptide sequence. In some embodiments, any synthetic signal peptide may comprise a pro-protein signal peptide (e.g., SEQ ID NOs. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75) fused to any native pre-protein signal peptide or portion thereof (e.g., pre-α-MF, SUC2 pre). In some embodiments, any synthetic signal sequence may comprise a pro-protein signal peptide (e.g., SEQ ID NOs. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75) fused to any synthetic pre-protein signal peptide (e.g., SEQ ID NO.s. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73) or portion thereof. Other examples of signal peptides that may be incorporated in their entirety or in part into a synthetic signaling peptide include but are not limited to, HSp150, PHO1, PHO5, SUC2, KILM1, GGP1, SUN, PLB, CRH, EXG, AGA2, HAS pre-pro, PIR1, XPR2 pre, XPR2 pre-pro, pGKL, SCW, and DSE.
In some embodiments, a method of generating an engineered yeast that expresses a recombinant polypeptide comprising a synthetic signal peptide is provided, the method comprising providing a yeast, contacting the yeast with a nucleic acid molecule encoding the recombinant polypeptide comprising the synthetic signal peptide, and culturing the yeast under conditions suitable to genetically modify the yeast to induce expression of the recombinant polypeptide, thereby creating an engineered yeast.
The yeast may be any strain of yeast, such as, but not limited to, Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger). In some embodiments, inducing expression of the recombinant polypeptide may be carried out via any expression system known to those skilled in the art. For example, in some embodiments, the method of generating an engineered yeast may comprise preparing a vector containing a nucleic acid (e.g., RNA, DNA) encoding the recombinant polypeptide, transporting the vector to the host yeast (“genetically modifying”), and culturing the yeast under effective conditions to express the recombinant polypeptide. As used herein, the term “vector” refers to a nucleotide molecule capable of transporting other nucleotides to which it has been linked. One exemplary type of vector is a “plasmid”, which represents a circular double stranded DNA loop into which additional DNA sections can be ligated. Another type of vector is a viral vector; wherein additional DNA sections can be ligated with the viral genome. Methods of introducing a DNA into yeast are known to those skilled in the art and may include a transformation method, a transfection method, an electroporation method, a nuclear injection method, or a carrier such as a liposome, micelle, skin cell, or a fusion method using protoplasts. A recombinant nucleic acid encoding the recombinant polypeptide may be obtained from any source using conventional techniques known to those skilled in the art, including isolation from genomic or cDNA libraries, amplification by PCR, or chemical synthesis.
In some embodiments, an engineered yeast may be cultured to induce growth of the yeast for a period of time in an environment effective to maintain the health of the yeast, thereby generating a desired amount of recombinant polypeptide comprising the synthetic signal peptide and payload protein. The culturing of yeast is common practice and well known in the art. In general, yeast can be grown in broth or agar in the presence of culture medium comprising bacteriological peptone, yeast extract, and glucose. Supplemental components such as amino acids, buffers, polysaccharides, and salts are sometimes used as well, depending on the strain and application. Engineered yeast may be grown at room temperature or, more effectively, at a temperature of up to about 30° C. to 37° C. Temperature may be used to control the growth of the yeast cells and to regulate the production of the desired recombinant polypeptide. Thus, in some embodiments, the yeast may be grown at a temperature from about 4° C. to about 50° C. The recited temperature range includes any temperature range within said range. Thus, in some embodiments, the yeast may be grown at a temperature from about 4° C. to about 40° C., from about 10° C. to about 50° C., from about 10° C., to about 45° C., from about 15° C., to about 45° C., from about 20° C. to about 45° C., from about 25° C. to about 45° C., from about 30° C. to about 50° C., from about 35° C. to about 50° C., from about 37° C. to about 50° C., from about 40° C. to about 50° C., or from about 45° C. to about 50° C. Similarly, the recited ranges include each and every individual temperature within said range. Thus, in some embodiments, the yeast may be grown at a temperature of about 4° C. In some embodiments, the yeast may be grown at a temperature of about 50° C. In some embodiments, the yeast may be grown at a temperature of about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C. Further, those skilled in the art will recognize that further modifications to the growth conditions may be necessary depending on the strain of yeast utilized and the recombinant polypeptide being produced. Such modifications are within the scope of the present application. In any case, secretion of a payload protein by the host yeast will result in its accumulation in the surrounding culture medium, where it may then be collected, isolated, and/or quantified. Through various intracellular mechanisms, the payload protein will be extracellularly secreted with or without some or all of the synthetic signal peptide to which it was fused.
In some embodiments, the proteins that may be produced by the engineered yeast include any protein. In some embodiments, the proteins that may be produced by the engineered yeast disclosed herein include, but are not limited to, maltose binding protein (MBP), trefoil factor, mucin, DNase, clotting or blood volumizing factors, insulin and insulin analogs, an incretin (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), EGFP, PDGF, HB-EGF, α1-antitrypsin, serum albumin, collagen, pepsinogen, tumor necrosis factor, streptokinase, glucagon, lepirudin, desirudin, hirudin, encallantide, IFN-α 2b, antigens and antibodies (e.g., anti-IL-6R Ab, anti-RSV ab, tetanus toxin fragment C, An-PEP, HIV-1 gp120 (intracellular), HIV-1 gp120 (secret), Bm86 tick gut glytoprotein, murine single-chain antibody, anti-TNF Ab, cancer antibodies, sHBsAg), enzymes (e.g., lysozyme, invertase, galactanase, isomaltase, lactase, chitiniase, xylanase, catalase, D-alanine carboxypeptidase, α-amylase, aspartic proteinase II, galactosidase, horseradish peroxidase, rasburicase, ocriplasmin, pancrelipase, alcohol dehydrogenase (I and II), phosphoglyserate kinase, GADPH, acid phosphatase), enzyme inhibitors (e.g., Kunitz protease inhibitor, tick anticoagulant protein, ghilanten, tPA Kringle type-2 domain), hormones (e.g., HGH, follicle stimulating hormone, human parathyroid hormone), vaccines (e.g., hepatitis vaccine (I), HPV vaccine), food processing products (e.g., brazzein, chymocin, beta-galactosidase), and cytokines.
In some embodiments, secretion of a payload protein by a yeast is increased by genetically modifying the yeast to express the payload protein as part of a recombinant polypeptide comprising a synthetic signal peptide as disclosed herein. Accordingly, in some embodiments, an engineered yeast may secrete about 10% to about 200% more of a payload protein than a yeast expressing a native signal peptide. In some embodiments, an engineered yeast may express about 10% to about 50% more, about 20% to about 70% more, about 30% to about 90% more, or about 50% to about 200% more of a payload protein. It is to be understood that any individual percentage of increased payload protein secretion is encompassed within the embodiments described herein. Accordingly, in some embodiments, the yeast may secrete about 10% more of a payload protein. In some embodiments, the yeast may secrete about 20% more of a payload protein. In some embodiments, the yeast may secrete about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% more of a payload protein, or any percentage falling within any of the recited percentages. Those of skill in the art would recognize that any change in growth condition during routine optimization for expression of a particular recombinant polypeptide of interest may also affect the amount of payload protein secreted by the engineered yeast. Accordingly, in some embodiment, an engineered yeast may secrete at least 10% more of a payload protein. Accordingly, in some embodiments, an engineered yeast may secrete about 10% more, about 100%, about 500% more, about 1000% more, or about 10,000% more of a payload protein compared to a yeast expressing a native signal peptide. In some embodiments, secretion is measured by measuring the concentration of the payload protein in the culture media in which the yeast was grown. The concentration may be normalized to optical density to account for variations in growth of the yeast. In some embodiments, secretion is measured by any method known to those skilled in the art for measuring payload protein concentration.
In some embodiments, the payload protein may be isolated from the culture medium in which the engineered yeast is grown using any methods known to those skilled in the art, such as precipitation from the medium, immunoaffinity chromatography, receptor affinity chromatography, or hydrophobic interaction chromatography. In some embodiments, the payload protein may be isolated by conventional chromatographic methods such as affinity chromatography, size-exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.
In some embodiments, a recombinant polypeptide may be designed to comprise a specific affinity peptide, tag, label, or chelate residue that is recognized by a specific binding partner or agent which may aid in isolation. In some embodiments, the recombinant polypeptide variants comprising the additional tag, label, or residue may then be cleaved to obtain the payload protein.
Synthetic Signal Peptides and Methods of their Use
In some embodiments, the various signal peptides disclosed herein may be utilized in yeast to deliver any payload protein to any environment. In some embodiments, an engineered yeast utilizing a signal peptide as disclosed herein may be used to deliver one or more of a therapeutic protein, diagnostic protein, or protein-based vaccine to a subject in need thereof. In some embodiments, the engineered yeast utilizing a signal peptide as disclosed herein may be used to deliver a payload protein to a specific organ or location within the subject, for example, to a subject's GI tract, skin, reproductive tract, or the like. In some embodiments the subject may be an animal, such as a companion animal (e.g., dog, cat, rodent, or the like). In some embodiments, the subject may be a livestock animal (e.g., cattle, sheep, horse, pig, goat, or the like). In some embodiments, the subject is a human.
In some embodiments, an engineered yeast may be used to deliver one or more of a protein-based herbicide, fungicide, bactericide, insecticide, nematicide, miticide, plant growth regulator, plant growth stimulant, or fertilizer in an agricultural environment, such as to crops or plants (such as seeds, roots, corn, tubers, bulbs, slip, rhizome, grass, or vines) or to a plant growth environment (such as topsoil, top dressing, compost, manure, water table, or hydroponic tank).
In some embodiments, an engineered yeast may be incorporated into a food product, such as bread, dairy, or fermented beverage, to deliver a therapeutic protein, diagnostic protein, protein-based vaccine, an anti-spoilage agent (e.g., bactericide or fungicide), protein-based flavoring agent, protein supplement, or an allergen degrader (e.g., gluten enzyme).
In some embodiments, an engineered yeast may be used to deliver any protein in any application or environment where fermentation is desired. Further specific uses are described herein below.
Therapeutic Compositions and Methods of their Use
The synthetic signal peptides and methods for their use, as disclosed herein, may be used to facilitate secretion of a payload protein expressed by a yeast. In some embodiments, the payload protein may have therapeutic efficacy and as such, may be used to treat a condition, disorder, or disease in a subject. Accordingly, in some embodiments, a method of treating a condition, disorder, or disease in a subject in need thereof in provided, the method comprising administering a composition comprising a therapeutically effective amount of a protein, wherein the protein is produced in an engineered yeast genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or both of a synthetic pre-protein signal and a synthetic pro-protein signal as disclosed herein. In some embodiments, administering may be performed via any route, such as oral or topical. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered topically.
In some embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic payload protein is provided, wherein the therapeutic payload protein is generated by an engineered yeast genetically modified with a nucleic acid molecule encoding a recombinant polypeptide comprising one or both of a synthetic pre- and pro-protein signal peptide, as disclosed in any aspect or embodiment herein. In some embodiments, the disease or condition may include, but is not limited to, an infection, an autoimmune disease, enzymatic deficiencies (including primary (congenital) enzymatic deficiency and enzymatic deficiencies secondary to functional gut disorders), diabetes, obesity, metabolic disorders, intestinal bacterial overgrowth, enteric infection, bacterial vaginosis, short bowel syndrome, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, Celiac disease, gluten intolerance, colitis, peptic ulcer, gastritis, polyps, hemorrhoids, cirrhosis, or a cancer.
In some embodiments, a composition comprising a therapeutic protein that is produced by any engineered yeast disclosed herein may be formulated for oral, topical, parenteral, or transdermal administration. These compositions may be in form of pill, tablet, capsule, microcapsule, powder, sachet, dragee, gel, liquid, suspension, solution, food product, cream or granule, and may further comprise one or more pharmaceutically acceptable excipients such as, but not limited to, carriers, solvents, co-solvents, emulsifiers, lubricants, disintegrants, binders, fillers, glidants, rheology agents, solubilizers, antimicrobials, antioxidants, preservatives, colorants, flavor agents, emollients, pH modifiers, and the like.
In some embodiments, food products may include, but are not limited to, a dairy product, a yoghurt, an ice cream, a milk-based drink, a milk-based garnish, a pudding, a milkshake, an ice tea, a fruit juice, a diet drink, a soda, a sports drink, a powdered drink mixture for dietary supplementation, an infant and baby food, a calcium-supplemented orange juice, a sauce or a soup.
In some embodiments, the engineered yeast may be utilized as a conduit for drug delivery to a subject. For example, engineered yeast may be orally administered to a subject to treat a condition, disorder, or disease, wherein the engineered yeast continues to produce and secrete the therapeutic protein within the subject, therefore providing a therapeutic benefit to the subject. Accordingly, in some embodiments, a method of treating a condition, disorder, or disease in a subject in need thereof is provided, the method comprising administering a therapeutically effective amount of engineered yeast as described herein, to the subject. In some embodiments, the therapeutically effective amount of engineered yeast may be orally administered to the subject. In some embodiments, the condition, disorder, or disease may include, but is not limited to, a GI disease or condition, a topical disease or condition, or a mucosal disease or condition. For example, the disease can be a viral (e.g. rotavirus), bacterial, fungal, or parasitic infection (such as, but not limited to intestinal bacterial overgrowth, bacterial vaginosis, an STI), an autoimmune disease (e.g., GBS), an enzymatic or vitamin deficiency (such as lactose intolerance, CSID, Celiac disease/gluten intolerance), a metabolic disorder such as diabetes, an inflammatory GI disease (e.g., irritable bowel syndrome, inflammatory bowel disease, colitis, gastritis, polyps), other GI condition or disease where healing/repair is required (e.g., peptic ulcer), an inflammatory skin condition (e.g. atopic dermatitis, diabetic ulcer), a wound, short bowel syndrome, hemorrhoids, cirrhosis, or a cancer. In some embodiments, administering may be performed via any route, such as oral or topical. The therapeutically effective amount of engineered yeast may be measured in colony forming units (CFUs) and may be any amount, such as from about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
In another embodiment, a pharmaceutical composition comprising an engineered yeast genetically modified with a nucleic acid molecule encoding a recombinant polypeptide comprising one or both of a synthetic pre- and pro-protein signal peptide, as disclosed in any aspect or embodiment herein, and a payload protein is provided.
In some embodiments, the composition comprises a Kluyveromyces yeast (e.g., K. lactis) genetically modified with a nucleic acid molecule encoding a recombinant polypeptide comprising one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21 fused directly or indirectly thereto and a payload protein. In some embodiments, the one or both signal peptides are fused directly to the payload protein. In some embodiments, the one or both signal peptides are fused indirectly to the payload protein via, for example, a linker peptide as provided for herein.
In some embodiments, the composition comprises a Pichia yeast (e.g., P. pastoris) genetically modified with a nucleic acid molecule encoding a recombinant polypeptide comprising one or both of a) a pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, 21 fused directly or indirectly thereto and a payload protein. In some embodiments, the one or both signal peptides are fused directly to the payload protein. In some embodiments, the one or both signal peptides are fused indirectly to the payload protein via, for example, a linker peptide as provided for herein.
In some embodiments, the composition comprises a Saccharomyces yeast (e.g. S. boulardii or S. cerevisiae) genetically modified with a nucleic acid molecule encoding a recombinant polypeptide comprising one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25 fused directly or indirectly thereto and a payload protein. In some embodiments, the one or both signal peptides are fused directly to the payload protein. In some embodiments, the one or both signal peptides are fused indirectly to the payload protein via, for example, a linker peptide as provided for herein.
In some embodiments, the composition comprises a Trichoderma yeast (e.g., T. reesei or T. viride) genetically modified with a nucleic acid molecule encoding recombinant polypeptide comprising one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38 fused directly or indirectly thereto and a payload protein. In some embodiments, the one or both signal peptides are fused directly to the payload protein. In some embodiments, the one or both signal peptides are fused indirectly to the payload protein via, for example, a linker peptide as provided for herein.
In some embodiments, the composition comprises an Aspergillus yeast (e.g., A. niger) genetically modified with a nucleic acid molecule encoding recombinant polypeptide comprising one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75 fused directly or indirectly thereto and a payload protein. In some embodiments, the one or both signal peptides are fused directly to the payload protein. In some embodiments, the one or both signal peptides are fused indirectly to the payload protein via, for example, a linker peptide as provided for herein.
In some embodiments, the disease or condition is an enzyme deficiency, and the payload protein is an enzyme.
In some embodiments, the disease or condition is congenital sucrose-isomaltase deficiency and the payload protein is one or both of invertase and isomaltase.
In some embodiments, the disease or condition is sucrose intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase. In some embodiments, the disease or condition is isomaltase intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase. In some embodiments, the disease or condition is one or both of sucrose and isomaltase intolerance secondary to a functional gut disorder and the payload protein is one or both of invertase and isomaltase.
In some embodiments, the disease or condition is one or more of gluten intolerance, refractory sprue, or Celiac disease and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide. In some embodiments, the disease or condition is gluten intolerance and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide. In some embodiments, the disease or condition is refractory sprue and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide. In some embodiments, the disease or condition is Celiac disease and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide.
In some embodiments, the disease or condition is pancreatitis or exocrine pancreatic insufficiency and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin. In some embodiments, the disease or condition is pancreatitis and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin. In some embodiments, the disease or condition is exocrine pancreatic insufficiency and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin.
In some embodiments, the disease or condition is enteropeptidase deficiency or enterokinase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase. In some embodiments, the disease or condition is enteropeptidase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase. In some embodiments, the disease or condition is enterokinase deficiency and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase.
In some embodiments, the disease or condition is small intestinal bacterial overgrowth, inflammatory bowel disease, irritable bowel syndrome, C. difficile infection, cystic fibrosis, necrotizing enterocolitis, and diabetes, and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is small intestinal bacterial overgrowth, and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is inflammatory bowel disease and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is irritable bowel syndrome and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is C. difficile infection and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is cystic fibrosis and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is necrotizing enterocolitis and the payload protein is intestinal alkaline phosphatase. In some embodiments, the disease or condition is diabetes and the payload protein is intestinal alkaline phosphatase.
In some embodiments, the disease or condition is short bowel syndrome and the payload protein is IGF-1, GLP-2, or a synthetic derivative of GLP-2. In some embodiments, the disease or condition is short bowel syndrome and the payload protein is IGF-1. In some embodiments, the disease or condition is short bowel syndrome and the payload protein is GLP-2. In some embodiments, the disease or condition is short bowel syndrome and the payload protein is a synthetic derivative of GLP-2.
In some embodiments, the disease or condition is lactose sensitivity or lactose intolerance and the payload protein is lactase. In some embodiments, the disease or condition is lactose sensitivity and the payload protein is lactase. In some embodiments, the disease or condition is lactose intolerance and the payload protein is lactase.
In some embodiments, the disease or condition is trehalose sensitivity or lactose intolerance and the payload protein is trehalase.
In some embodiments, the disease or condition is maltose sensitivity or lactose intolerance and the payload protein is maltase. In some embodiments, the disease or condition is maltose sensitivity and the payload protein is maltase. In some embodiments, the disease or condition is lactose intolerance and the payload protein is maltase.
In some embodiments, the disease or condition is pernicious anemia and the payload protein is intrinsic factor.
In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is lysozyme, nisin, a defensin, magainin, cateslytin, or any combination thereof. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is lysozyme. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is nisin. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is a defensing. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is magainin. In some embodiments, the disease or condition is bacterial overgrowth and the payload protein is cateslytin.
In some embodiments, the disease or condition is type 1 or type 2 diabetes mellitus and the payload protein is insulin, or an incretin. In some embodiments, the disease or condition is type 1 diabetes mellitus and the payload protein is insulin, or an incretin. In some embodiments, the disease or condition is type 1 diabetes mellitus and the payload protein is insulin. In some embodiments, the disease or condition is type 1 diabetes mellitus and the payload protein is an incretin. In some embodiments, the disease or condition is type 2 diabetes mellitus and the payload protein is insulin, or an incretin. In some embodiments, the disease or condition is type 2 diabetes mellitus and the payload protein is insulin. In some embodiments, the disease or condition is type 2 diabetes mellitus and the payload protein is an incretin.
In some embodiments, the disease or condition has an inflammatory component and the payload protein is IL-10, IL-22, TGFβ, or any combination thereof.
Methods of Treating Invertase/Sucrase and/or Isomaltase Deficiency
An engineered yeast may be used, for example, to treat an enzyme deficiency such as a deficiency of invertase and/or isomaltase. Accordingly, in some embodiments a method of treating a sucrase/invertase and/or isomaltase deficiency in a subject in need thereof is provided, the method comprising orally administering to the subject one or both of 1) a therapeutically effective amount of an engineered yeast genetically modified to express a first recombinant polypeptide comprising invertase (or a pro-drug or active variant thereof) and a first synthetic signal peptide and 2) a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising isomaltase (or a pro-drug or active variant thereof) and a second synthetic signal peptide, thereby treating the invertase and/or isomaltase deficiency. In some embodiments, the first and second synthetic signal peptide independently comprise one or both of a) a pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger). In some embodiments, the invertase and/or isomaltase deficiency may be secondary to a functional gut disorder, such as, but not limited to, irritable bowel syndrome, functional dyspepsia, functional vomiting, functional abdominal pain, functional constipation, and/or functional diarrhea.
In some embodiments, a method of treating a sucrase/invertase and/or isomaltase deficiency is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with one or more of 1) a nucleic acid encoding a recombinant polypeptide comprising invertase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21 and 2) a nucleic acid encoding a recombinant polypeptide comprising isomaltase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby treating the deficiency.
In some embodiments, a method of treating a sucrase/invertase and/or isomaltase deficiency is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with one or both of 1) a nucleic acid encoding a recombinant polypeptide comprising invertase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21 and 2) a nucleic acid encoding a recombinant polypeptide comprising isomaltase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby treating the deficiency.
In some embodiments, a method of treating a sucrase/invertase and/or isomaltase deficiency is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with one or both of 1) a nucleic acid encoding a recombinant polypeptide comprising isomaltase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25 and 2) a nucleic acid encoding a recombinant polypeptide comprising invertase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating the deficiency.
In some embodiments, a method of treating a sucrase/invertase and/or isomaltase deficiency is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with one or both of 1) a nucleic acid encoding a recombinant polypeptide comprising isomaltase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38 and 2) a nucleic acid encoding a recombinant polypeptide comprising invertase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the deficiency.
In some embodiments, a method of treating a sucrase/invertase and/or isomaltase deficiency is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with one or both of 1) a nucleic acid encoding a recombinant polypeptide comprising isomaltase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75 and 2) a nucleic acid encoding a recombinant polypeptide comprising invertase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the deficiency.
In some embodiments, the sucrase/invertase and/or isomaltase deficiency may be, for example, congenital sucrase-isomaltase deficiency. In any embodiment where a subject has both a sucrase/invertase and isomaltase deficiency and it is desired to administer engineered yeast to express both enzymes, the same yeast strain may be used to express both enzymes or one yeast strain may be used to express invertase and another yeast strain may be used to express isomaltase. In some embodiments, administration of both enzymes is performed utilizing one yeast strain to express both enzymes. In some embodiments, administration of both enzymes is performed utilizing one yeast strain to express invertase and another yeast strain to express isomaltase.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
In some embodiments, a method of treating a lactase deficiency or lactose-intolerance in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising lactase (or a pro-drug or active variant thereof) and a synthetic signal peptide, thereby treating lactase deficiency or lactose-intolerance. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of treating a lactase deficiency/lactose-intolerance is provided, the method comprising administering to a subject in need thereof a Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising lactase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby treating the deficiency.
In some embodiments, a method of treating a lactase deficiency/lactose-intolerance is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising lactase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby treating the deficiency.
In some embodiments, a method of treating a lactase deficiency/lactose-intolerance is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising lactase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating the deficiency.
In some embodiments, a method of treating a lactase deficiency/lactose-intolerance is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising lactase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the deficiency.
In some embodiments, a method of treating a lactase deficiency/lactose-intolerance is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising lactase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the deficiency.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
In some embodiments, a method of treating a pancreatic disorder, such as pancreatitis or exocrine pancreatic insufficiency, in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin and a synthetic signal peptide, thereby treating the disorder. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger). In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising triacylglycerol lipase and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency. In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising colipase and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency. In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising alpha-amylase and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency. In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising trypsin and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency. In some embodiments, the engineered yeast is genetically modified to express a recombinant polypeptide comprising chymotrypsin and a synthetic signal peptide as provided for herein, and is effective for treating one or both of pancreatitis or exocrine pancreatic insufficiency.
In some embodiments, a method of treating pancreatitis or exocrine pancreatic insufficiency is provided, the method comprising administering to a subject in need thereof a Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VII or SEQ ID NO. 20 or 21, thereby treating the disorder.
In some embodiments, a method of treating pancreatitis or exocrine pancreatic insufficiency is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby treating the disorder.
In some embodiments, a method of treating pancreatitis or exocrine pancreatic insufficiency is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating the disorder.
In some embodiments, a method of treating pancreatitis or exocrine pancreatic insufficiency is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the disorder.
In some embodiments, a method of treating pancreatitis or exocrine pancreatic insufficiency is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the disorder.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
In some embodiments, a method of treating a deficiency of one or more of Aspergillus niger prolyl endoprotease (An-PEP), Myxococcus xanthus prolyl endopeptidase (Mx-PEP), Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide (or a pro-drug or active variant thereof) and a synthetic signal peptide, thereby treating the deficiency. In some embodiments, the synthetic signal peptide comprises one or both of a) a pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the yeast strain is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger). In some embodiments, the recombinant polypeptide comprises An-PEP and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises Mx-PEP and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises Aspergillus tubigensis prolyl endopeptidase and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises subtilisin and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises sedolisin and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue. In some embodiments, the recombinant polypeptide comprises larozotide and a synthetic signal peptide as provided for herein, and the engineered yeast is effective to treat Celiac Disease, Gluten Intolerance, or refractory sprue.
In some embodiments, a method of treating one or more of Celiac Disease, gluten intolerance, and refractory sprue is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20, 21, thereby treating the disease or disorder.
In some embodiments, a method of treating one or more of Celiac Disease, gluten intolerance, and refractory sprue is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21 or Var. Seq. 6, thereby treating the disease or disorder.
In some embodiments, a method of treating one or more of Celiac Disease, gluten intolerance, and refractory sprue is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, 25, thereby treating the disease or disorder.
In some embodiments, a method of treating one or more of Celiac Disease, gluten intolerance, and refractory sprue is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the disease or disorder.
In some embodiments, a method of treating one or more of Celiac Disease, gluten intolerance, and refractory sprue is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larozotide and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the disease or disorder.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
Enterokinase or enteropeptidase deficiency is an autosomal recessive disorder characterized by severe protein malabsorption in early infancy and may be treated by an engineered yeast according to the present disclosure. Accordingly, in some embodiments, a method of treating enterokinase/enteropeptidase deficiency in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or both of enteropeptidase (enterokinase) and proenteropeptidase and a synthetic signal peptide, thereby treating the disorder. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of treating enterokinase or enteropeptidase deficiency is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of enteropeptidase/enterokinase and proenteropeptidase and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby treating the disorder.
In some embodiments, a method of treating enterokinase or enteropeptidase deficiency is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of enteropeptidase/enterokinase and proenteropeptidase and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby treating the disorder.
In some embodiments, a method of treating enterokinase or enteropeptidase deficiency is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of enteropeptidase/enterokinase and proenteropeptidase and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, or, thereby treating the disorder.
In some embodiments, a method of treating enterokinase or enteropeptidase deficiency is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of enteropeptidase/enterokinase and proenteropeptidase and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the disorder.
In some embodiments, a method of treating enterokinase or enteropeptidase deficiency is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of enteropeptidase/enterokinase and proenteropeptidase and 2) one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the disorder.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
In some embodiments, a method of treating bacterial infection or bacterial overgrowth in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising 1) one or both of lysozyme and intestinal alkaline phosphatase and 2) a synthetic signal peptide, thereby treating the infection or overgrowth. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger). In some embodiments, the bacterial infection or overgrowth may include, but not be limited to, a small intestine bacterial overgrowth, which may be associated with diabetes, a C. difficile infection, and intestinal bacterial overgrowth associated with cystic fibrosis. In some embodiments, the bacterial infection may be caused by be any gram-positive or gram-negative bacteria, such as, but not limited to, an infection of Escherichia Coli (E. Coli), Clostridioides difficile, P. aeruginosa, Shigella, Salmonella, Vibrio cholera, or cryptosporidium.
In some embodiments, a method of treating a bacterial overgrowth or infection is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of lysozyme and intestinal alkaline phosphatase and 2) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby treating the infection or overgrowth.
In some embodiments, a method of treating a bacterial overgrowth or infection is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of lysozyme and intestinal alkaline phosphatase and 2) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby treating the infection or overgrowth.
In some embodiments, a method of treating a bacterial overgrowth or infection is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of lysozyme and intestinal alkaline phosphatase and 2) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating the infection or overgrowth.
In some embodiments, a method of treating a bacterial overgrowth or infection is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of lysozyme and intestinal alkaline phosphatase and 2) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the infection or overgrowth.
In some embodiments, a method of treating a bacterial overgrowth or infection is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising 1) one or both of lysozyme and intestinal alkaline phosphatase and 2) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the infection or overgrowth.
In some embodiments, other antibacterial proteins that may be produced by an engineered yeast and therefore provide treatment for bacterial overgrowth or infection in a subject include human beta defensins, peptide antimicrobials of animal origin (e.g., magainin, dermaseptin, cateslytin), and peptide antimicrobials of microbe origin (e.g., nisin, sakacin). In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
In some embodiments, the method of treating a bacterial infection with an engineered yeast genetically modified to express lysozyme, as described herein, may further comprise administering an antibacterial agent in combination with the engineered yeast. For example, a bacterial infection may be treated by administering a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising a synthetic signal peptide and lysozyme and a therapeutically effective amount of an antibacterial agent. In some embodiments, the antibacterial agent is selected from the group comprising quinupristin, piperacillin, penicillin, clarithromycin, nitrofurantoin, ciprofloxacin, telithromycin, metronidazole, levofloxacin, erythromycin, theophylline, gemifloxacin, tetracycline, azithromycin, delafloxacin, eravacycline, moxifloxacin, dalbavancin, amoxicillin, fidaxomicin, tigecycline, ceftriaxone, minocycline, rifapentine, clindamycin, ceftazidime, oritayancin, norfloxacin, doxycycline, cefuroxime, tobramycin, ceftibuten, gentamicin, cefotaxime, vancomycin, telavancin, daptomycin, cephalexin, fofomycin, tedizolid, aztreonam, nafcillin, phenytoin, ertapenem, cefazolin, isoniazid, doripenem, rifabutin, meropenem, linezolid, oflaxacin, cefoxitin, oxacillin, warfarin, neomycin, rifampin, cefepime, and digoxin. In some embodiments, the antibacterial agent can be administered by any route, such as oral, topical, intranasal, mucosal, otic, parenteral, or the like.
In some embodiments, a method of treating inflammatory gastrointestinal disorders in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising intestinal alkaline phosphatase and a synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger). In some embodiments, the inflammatory gastrointestinal disorder is selected from the group including, but not limited to, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and necrotizing enterocolitis.
In some embodiments, a method for treating an inflammatory gastrointestinal disorder is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intestinal alkaline phosphatase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby treating the disorder. In some embodiments, the inflammatory gastrointestinal disorder is selected from the group comprising IBS, IBD, and necrotizing enterocolitis.
In some embodiments, a method for treating an inflammatory gastrointestinal disorder is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intestinal alkaline phosphatase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby treating the disorder. In some embodiments, the inflammatory gastrointestinal disorder is selected from the group comprising IBS, IBD, and necrotizing enterocolitis.
In some embodiments, a method for treating an inflammatory gastrointestinal disorder is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intestinal alkaline phosphatase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating the disorder. In some embodiments, the inflammatory gastrointestinal disorder is selected from the group comprising IBS, IBD, and necrotizing enterocolitis.
In some embodiments, a method for treating an inflammatory gastrointestinal disorder is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intestinal alkaline phosphatase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the disorder. In some embodiments, the inflammatory gastrointestinal disorder is selected from the group comprising IBS, IBD, and necrotizing enterocolitis.
In some embodiments, a method for treating an inflammatory gastrointestinal disorder is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intestinal alkaline phosphatase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the disorder. In some embodiments, the inflammatory gastrointestinal disorder is selected from the group comprising IBS, IBD, and necrotizing enterocolitis.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
An engineered yeast may be used to treat an insulin deficiency or disorder, such as type 1 and type 2 diabetes mellitus. Accordingly, in some embodiments, a method of treating type 1 or type 2 diabetes mellitus in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising insulin (or a peptide analog or pro-drug thereof) and a synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising insulin (or a peptide analog or pro-drug thereof) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby treating the deficiency or disease.
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising insulin (or a peptide analog or pro-drug thereof) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby treating the deficiency or disease.
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising insulin (or a peptide analog or pro-drug thereof) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, 25, thereby treating the deficiency or disease.
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising insulin (or a peptide analog or pro-drug thereof) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the deficiency or disease.
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising insulin (or a peptide analog or pro-drug thereof) and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the deficiency or disease.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
In some embodiments, a method of treating type 1 or type 2 diabetes mellitus in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising an incretin and a synthetic signal peptide, thereby treating the type 1 or type 2 diabetes mellitus. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger). In some embodiments, the incretin is selected from the group including, but not limited to, GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin, diaglutide, exenatide, liraglutide, semaglutide, sitagliptin, saxagliptin, alogliptin, linagliptin, and GIP.
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising an incretin and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby treating the deficiency or disease. In some embodiments, the incretin is selected from the group including, but not limited to, GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin, diaglutide, exenatide, liraglutide, semaglutide, sitagliptin, saxagliptin, alogliptin, linagliptin, and GIP.
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising an incretin and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby treating the deficiency or disease. In some embodiments, the incretin is selected from the group including, but not limited to, GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin, diaglutide, exenatide, liraglutide, semaglutide, sitagliptin, saxagliptin, alogliptin, linagliptin, and GIP.
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising an incretin and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, 25, thereby treating the deficiency or disease. In some embodiments, the incretin is selected from the group including, but not limited to, GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin, diaglutide, exenatide, liraglutide, semaglutide, sitagliptin, saxagliptin, alogliptin, linagliptin, and GIP).
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising an incretin and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the deficiency or disease. In some embodiments, the incretin is selected from the group including, but not limited to, GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin, diaglutide, exenatide, liraglutide, semaglutide, sitagliptin, saxagliptin, alogliptin, linagliptin, and GIP.
In some embodiments, a method of treating an insulin deficiency/diabetes is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising an incretin and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the deficiency or disease. In some embodiments, the incretin is selected from the group including, but not limited to, GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin, diaglutide, exenatide, liraglutide, semaglutide, sitagliptin, saxagliptin, alogliptin, linagliptin, and GIP.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
An engineered yeast may be used to promote healing and repair of GI epithelium, for example, as caused by any disease or condition such as IBD or IBS, through the production of trefoil factors (e.g., TFF1/2/3) or IGF-1.
Accordingly, in some embodiments, a method of promoting growth and repair in GI endothelium in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of TFF1, TFF2, TFF3, or IGF-1 and synthetic signal peptide, thereby promoting growth and repair in GI endothelium. In some embodiments, the synthetic signal peptide comprises one or both of a) a pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of promoting GI growth and repair is provided, the method comprising administering Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising TFF1, TFF2, TFF3, or IGF-1 and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby promoting GI growth and repair.
In some embodiments, a method of promoting GI growth and repair is provided, the method comprising administering Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising TFF1, TFF2, TFF3, or IGF-1 and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby promoting GI growth and repair.
In some embodiments, a method of promoting GI growth and repair is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising TFF1, TFF2, TFF3, or IGF-1 and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, 25, thereby promoting GI growth and repair.
In some embodiments, a method of promoting GI growth and repair is provided, the method comprising administering a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising TFF1, TFF2, TFF3, or IGF-1 and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby promoting GI growth and repair.
In some embodiments, a method of promoting GI growth and repair is provided, the method comprising administering an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising TFF1, TFF2, TFF3, or IGF-1 and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby promoting GI growth and repair.
In any embodiment, growth and/or repair of GI epithelium may be in the context of a condition or disease such as short bowel syndrome, IBS, IBD, or any other disease where the GI epithelium is damaged or dysfunctional. In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
An engineered yeast may be used to treat short bowel syndrome. Accordingly, in some embodiments, a method of treating short bowel syndrome in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising IGF-1, GLP-2 or any synthetic analog or prodrug thereof and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) a pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of treating short bowel syndrome is provided, the method comprising administering Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising IGF-1, GLP-2 or any synthetic analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20, 21, thereby treating short bowel syndrome.
In some embodiments, a method treating short bowel syndrome is provided, the method comprising administering Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising IGF-1 or an analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby treating short bowel syndrome.
In some embodiments, a method of treating short bowel syndrome is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising IGF-1, GLP-2 or any synthetic analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, 25, thereby treating short bowel syndrome.
In some embodiments, a method of treating short bowel syndrome is provided, the method comprising administering a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising IGF-1, GLP-2 or any synthetic analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating short bowel syndrome.
In some embodiments, a method of treating short bowel syndrome is provided, the method comprising administering an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising IGF-1, GLP-2 or any synthetic analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating short bowel syndrome.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
Trehalase deficiency is a metabolic condition where the body lacks the enzyme trehalase and is therefore unable to convert trehalose into glucose. Accordingly, in some embodiments, a method of treating a trehalase deficiency in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising trehalase (or a pro-drug or active variant thereof) and a synthetic signal peptide, thereby treating the deficiency. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method for treating trehalose sensitivity is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising trehalase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby treating the trehalose sensitivity.
In some embodiments, a method for treating trehalose sensitivity is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising trehalase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby treating the trehalose sensitivity.
In some embodiments, a method of treating trehalose sensitivity is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising trehalase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating the trehalose sensitivity.
In some embodiments, a method of treating trehalose sensitivity is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising trehalase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the trehalose sensitivity.
In some embodiments, a method of treating trehalose sensitivity is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising trehalase and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the trehalose sensitivity.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
Pernicious anemia is a rare blood disorder characterized by the inability of the body to properly utilize vitamin B12, resulting from the lack of the gastric protein intrinsic factor, without which B12 cannot be absorbed. Accordingly, in some embodiments, a method of treating pernicious anemia in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising intrinsic factor (or a pro-drug or active variant thereof) and a synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of treating pernicious anemia is provided, the method comprising administering to a subject in need thereof Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intrinsic factor and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby treating pernicious anemia.
In some embodiments, a method of treating pernicious anemia is provided, the method comprising administering to a subject in need thereof Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intrinsic factor and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20 or 21, thereby treating pernicious anemia.
In some embodiments, a method of treating pernicious anemia is provided, the method comprising administering to a subject in need thereof a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intrinsic factor and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating pernicious anemia.
In some embodiments, a method of treating pernicious anemia is provided, the method comprising administering to a subject in need thereof a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intrinsic factor and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating pernicious anemia.
In some embodiments, a method of treating pernicious anemia is provided, the method comprising administering to a subject in need thereof an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising intrinsic factor and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating pernicious anemia.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
An engineered yeast may be used to produce pro-repair cytokines such as IL-10, IL-22, and/or TGFβ, which may be suitable for treating a variety of diseases and conditions. Further, engineered yeast may be used to produce anti-TNFα antibodies or fragments of anti-TNFα antibodies. Oral administration of IL-10, IL-22, TGFβ and/or anti-TNFα antibodies or fragments thereof may be beneficial for treating and repairing damage caused by inflammatory GI conditions, such as IBS, IBD, and the like. In some embodiments, an engineered yeast genetically modified to express IL-10 may be orally administered to a subject to treat Crohn's disease or inhibit tumor metastasis. Accordingly, in some embodiments, a method of treating an inflammatory condition in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of IL-10, IL-22, TGFβ, and anti-TNFα antibodies or fragments thereof, or an analog or prodrug thereof and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of treating inflammation is provided, the method comprising administering Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of IL-10, IL-22, TGFβ, and anti-TNFα antibodies or fragments thereof, or an analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby treating the inflammation.
In some embodiments, a method of treating inflammation is provided, the method comprising administering Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of IL-10, IL-22, TGFβ, and anti-TNFα antibodies or fragments thereof, or an analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby treating the inflammation.
In some embodiments, a method of treating inflammation is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of IL-10, IL-22, TGFβ, and anti-TNFα antibodies or fragments thereof, or an analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating the inflammation.
In some embodiments, a method of treating inflammation is provided, the method comprising administering a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of IL-10, IL-22, TGFβ, and anti-TNFα antibodies or fragments thereof, or an analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the inflammation.
In some embodiments, a method of treating inflammation is provided, the method comprising administering an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of IL-10, IL-22, TGFβ, and anti-TNFα antibodies or fragments thereof, or an analog or prodrug thereof and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the inflammation.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
An engineered yeast may be used for treating a variety of cancers, for example, but not limited to, cancers of the GI tract. Accordingly, in some embodiments, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of an anti-cancer therapeutic and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of treating cancer is provided, the method comprising administering Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of an anti-cancer therapeutic and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby treating the inflammation.
In some embodiments, a method of treating cancer is provided, the method comprising administering Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of an anti-cancer therapeutic and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby treating the inflammation.
In some embodiments, a method of treating cancer is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more an anti-cancer therapeutic and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby treating the inflammation.
In some embodiments, a method of treating cancer is provided, the method comprising administering a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of an anti-cancer therapeutic and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby treating the inflammation.
In some embodiments, a method of treating cancer is provided, the method comprising administering an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of an anti-cancer therapeutic and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby treating the inflammation.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
An engineered yeast may be used to induce the release of the peptide hormone cholecystokinin (CCK, also known as pancreozymin), which has important roles in digestion and satiety. Oral administration of luminal CCK-releasing factor (LCRF) may be beneficial for promoting appetite suppression, delaying of gastric emptying, and/or inducing pancreatic secretion. Other proteins that exhibit these same functions include casein and soy proteins. Thus, administration of LCRF, casein, and/or soy proteins may be useful in the treatment of several digestive disorders and obesity through i) the suppression of appetite and ii) the promotion of digestion. In some embodiments, an engineered yeast genetically modified to express LCRF, casein, and/or soy proteins may be orally administered to a subject to promote appetite suppression. Accordingly, in some embodiments, a method of promoting appetite suppression in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising LCRF and synthetic signal peptide. In some embodiments, the recombinant polypeptide comprises casein. In some embodiments, the recombinant polypeptide comprises soy proteins. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of promoting appetite suppression is provided, the method comprising administering Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby promoting appetite suppression.
In some embodiments, a method of promoting appetite suppression is provided, the method comprising administering Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby promoting appetite suppression.
In some embodiments, a method of promoting appetite suppression is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) apro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby promoting appetite suppression.
In some embodiments, a method of promoting appetite suppression is provided, the method comprising administering a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby promoting appetite suppression.
In some embodiments, a method of promoting appetite suppression is provided, the method comprising administering an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby promoting appetite suppression.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
An engineered yeast may be used to induce the release of the peptide hormone cholecystokinin (CCK, also known as pancreozymin), which has important roles in digestion and satiety. Oral administration of luminal CCK-releasing factor (LCRF) may be beneficial for promoting appetite suppression, delaying of gastric emptying, and/or inducing pancreatic secretion. Other proteins that exhibit these same functions include casein and soy proteins. Thus, administration of LCRF, casein, and/or soy proteins may be useful in the treatment of several digestive disorders and obesity through i) the suppression of appetite and ii) the promotion of digestion. In some embodiments, an engineered yeast genetically modified to express LCRF, casein, and/or soy proteins may be orally administered to a subject to promote appetite suppression. Accordingly, in some embodiments, a method of delaying of gastric emptying in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising LCRF and synthetic signal peptide. In some embodiments, the recombinant polypeptide comprises casein. In some embodiments, the recombinant polypeptide comprises soy proteins. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of delaying of gastric emptying is provided, the method comprising administering Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby delaying gastric emptying.
In some embodiments, a method of delaying of gastric emptying is provided, the method comprising administering Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby delaying gastric emptying.
In some embodiments, a method of delaying of gastric emptying is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) apro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby delaying gastric emptying.
In some embodiments, a method of delaying gastric emptying is provided, the method comprising administering a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby delaying gastric emptying.
In some embodiments, a method of delaying of gastric emptying is provided, the method comprising administering an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby delaying gastric emptying.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
An engineered yeast may be used to induce the release of the peptide hormone cholecystokinin (CCK, also known as pancreozymin), which has important roles in digestion and satiety. Oral administration of luminal CCK-releasing factor (LCRF) may be beneficial for promoting appetite suppression, delaying of gastric emptying, and/or inducing pancreatic secretion. Other proteins that exhibit these same functions include casein and soy proteins. Thus, administration of LCRF, casein, and/or soy proteins may be useful in the treatment of several digestive disorders and obesity through i) the suppression of appetite and ii) the promotion of digestion. In some embodiments, an engineered yeast genetically modified to express LCRF, casein, and/or soy proteins may be orally administered to a subject to promote appetite suppression. Accordingly, in some embodiments, a method of inducing pancreatic secretion in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising LCRF and synthetic signal peptide. In some embodiments, the recombinant polypeptide comprises casein. In some embodiments, the recombinant polypeptide comprises soy proteins. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of inducing pancreatic secretion is provided, the method comprising administering Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby inducing pancreatic secretion.
In some embodiments, a method of inducing pancreatic secretion is provided, the method comprising administering Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby inducing pancreatic secretion.
In some embodiments, a method of inducing pancreatic secretion is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) apro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby inducing pancreatic secretion.
In some embodiments, a method of inducing pancreatic secretion is provided, the method comprising administering a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby inducing pancreatic secretion.
In some embodiments, a method of inducing pancreatic secretion is provided, the method comprising administering an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising LCRF and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby inducing pancreatic secretion.
In some embodiments, administering may be performed via any route. In some embodiments, the route of administration is oral or topical. The therapeutically effective amount of engineered yeast may be, for example, about 100 CFUs to 1020 CFUs, about 103 to 1015 CFUs, 104 to 1010 CFUs, or about 102 to about 108 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs to about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 103 to about 1015 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is from about 100 CFUs, about 103 CFUs, or about 104 CFUs to about 108 CFUs, about 1010 CFUs, about 1015 CFUs, or about 1020 CFUs. In some embodiments, the therapeutically effective amount of engineered yeast is any amount of CFU that falls within any of the above ranges.
In any method of administering the engineered yeast as a therapeutic, the engineered yeast may be incorporated into a composition suitable for oral administration to the subject. Accordingly, in some embodiments, a composition is provided, the composition comprising an engineered yeast as provided for herein. Advantageously, the engineered yeast, as disclosed herein, retain activity even after lyophilization and/or freeze-drying providing a particularly shelf-stable form for incorporating into pharmaceutical products, such as those for reconstitution prior to consumption. Accordingly, in some embodiments, the engineered yeast in the pharmaceutical composition can be provided in a lyophilized or freeze-dried form. An oral composition comprising an engineered yeast, as disclosed herein, may be in the form of a pill, tablet, capsule, microcapsule, powder, sachet, dragee, gel, liquid, suspension, solution, food product, cream or granule. In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutically acceptable excipient is selected from the group including, but not limited to, carriers, solvents, co-solvents, emulsifiers, lubricants, disintegrants, binders, fillers, glidants, rheology agents, solubilizers, antimicrobials, antioxidants, preservatives, colorants, flavor agents, emollients, pH modifiers, and the like.
In some embodiments, food products may include, but are not limited to, a dairy product, a yoghurt, an ice cream, a milk-based drink, a milk-based garnish, a pudding, a milkshake, an ice tea, a fruit juice, a diet drink, a soda, a sports drink, a powdered drink mixture for dietary supplementation, an infant and baby food, a calcium-supplemented orange juice, a sauce or a soup.
Agricultural Compositions and Methods of their Use
An engineered yeast may be used to produce agricultural payload proteins such as, but not limited to, decomposition enzymes (e.g., cellulose), soil and other agricultural enzymes (e.g., lipases, proteases, polymerases, amylases, peroxidases, catalases, beta glucosidase, FDA hydrolysis, amidase, urease, phosphatase, sulfatase) fungicides (e.g., chitinase, chitin-binding proteins, cyclophilin-like proteins, defensins, lipid transfer proteins, miraculin-like proteins, nucleases, thaumatin-like proteins, and the like), insecticides (e.g., Vip1, Vip2, Vip3, Cry proteins, and the like), plant activators (e.g., branched-β-glucans, chitin oligomers, pectolytic enzymes, elicitor activity independent from enzyme activity (e.g. endoxylanase, elicitins, PaNie), avr gene products (e.g., AVR4, AVR9), viral proteins (e.g., vial coat protein, Harpins), flagellin, protein or peptide toxin (e.g., victorin), glycoproteins, glycopeptide fragments of invertase, syringolids, Nod factors (lipochitoolingo-saccharides), FACs (fatty acid amino acid conjugates), ergosterol, bacterial toxins (e.g., coronatine), and sphinganine analogue mycotoxins (e.g., fumonisin B1), which may be suitable for treating a variety of diseases and conditions. Application of one or more of the above described agricultural payload proteins to an agricultural environment, such as a crop, garden, or the like, may be beneficial for promoting soil and plant health. Accordingly, in some embodiments, a method of promoting soil and/or plant health is provided, the method comprising applying to the soil or plant an effective amount of an engineered yeast genetically modified to express a recombinant polypeptide comprising one or more of an agricultural payload protein and synthetic signal peptide. In some embodiments, the synthetic signal peptide comprises one or both of a) an pre-protein amino acid sequence of Formula II, Formula III, Formula IV, Formula V, Formula IX, Formula XIII or SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; and b) a pro-protein amino acid sequence of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, Formula XV or SEQ ID NO. 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the engineered yeast may be any strain as disclosed herein. In some embodiments, the engineered yeast is selected from the group comprising Kluyveromyces (e.g., K. lactis), Pichia (e.g., P. pastoris), Saccharomyces (e.g., S. cerevisiae, S. boulardii), Trichoderma (e.g., T. reesei, T. viride), and Aspergillus (e.g., A. niger).
In some embodiments, a method of promoting soil and/or plant health is provided, the method comprising administering Kluyveromyces yeast (e.g., K. lactis), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of an agricultural payload protein as provided for herein and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula I or SEQ ID NO. 1 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 20 or 21, thereby promoting soil and/or plant health.
In some embodiments, a method of promoting soil and/or plant health is provided, the method comprising administering Pichia yeast (e.g., P. pastoris), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of an agricultural payload protein as provided for herein and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula II or SEQ ID NO. 2, 3, 4, 5, 6, or 7 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI or SEQ ID NO. 17, 20, or 21, thereby promoting soil and/or plant health.
In some embodiments, a method of promoting soil and/or plant health is provided, the method comprising administering a Saccharomyces yeast (e.g., S. cerevisiae or S. boulardii), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or an agricultural payload protein as provided for herein and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO. 8, 9, 10, 11, 12, 13, 14, 15, or 16 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO. 18, 19, 20, 21, 22, 23, 24, or 25, thereby promoting soil and/or plant health.
In some embodiments, a method of promoting soil and/or plant health is provided, the method comprising administering a Trichoderma yeast (e.g., T. reesei or T. viride), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of an agricultural payload protein as provided for herein and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula IX or SEQ ID NO. 31, 32, or 33 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula X, Formula XI, or SEQ ID NO. 34, 35, 36, 37, or 38, thereby promoting soil and/or plant health.
In some embodiments, a method of promoting soil and/or plant health is provided, the method comprising administering an Aspergillus yeast (e.g., A. niger), genetically modified with a nucleic acid encoding a recombinant polypeptide comprising one or more of an agricultural payload protein as provided for herein and one or both of a) a pre-protein signal peptide comprising an amino acid sequence of Formula XIII or SEQ ID NO. 70, 71, 72, or 73 and b) a pro-protein signal peptide comprising an amino acid sequence of Formula XIV, Formula XV, or SEQ ID NO. 74 or 75, thereby promoting soil and/or plant health.
In some embodiments, administering may be performed via any route. In some embodiments, the composition is sprayed onto the soil and/or plants. The agriculturally effective amount of engineered yeast may be any amount necessary to result in the desired beneficial effect to soil and or plant health.
In some embodiments, the following embodiments are provided:
1. A pre-protein signal peptide comprising an amino acid sequence selected from the group consisting of Formula I, II, III, IV, V, IX, and XIII wherein Formula I is given by:
A1-(A2)w-A3-(A4)x-(A5)y-A6-A7-A8-A9-A10-(A11)z (Formula I)
wherein:
B1-(B2)u-(B3)v-(B4)w-(B5)x-(B6)y-B7-B8-B9-B10-(B11)z (Formula II)
wherein:
C1-(C2)r-(C3)t-(C4)u-[(C5)v-(C6)w]x-(C7)y-(C8)z-C9-C10-C11-[C12-C13]a (Formula III)
wherein:
D1-(D2)q-(D3)r-(D4)t-(D5)u-[(D6)v-(D7)x-(D8)w-(D9)y]z-D10-D11-D12-[D13-D14]a (Formula IV)
wherein:
E1-[(E2)i-(E3)j-(E4)q]r-(E5)t-(E6)u-(E7)v-[(E8)w-(E9)x]y-(E10)z-E11-E12-E13-[E14-E15]a (Formula V)
wherein:
F1-(F2)v-(F3)w-[(F4)x-(F5)y]z-F6-F7-F8-[F9-F10]a (Formula IX)
wherein:
L1-(L2)x-[(L3)a-(L4)a]y-[(L5)a-(L6)a-(L7)a]z-(L8)a-(L9)a-(L10)a-(L11)a-(L12)a (Formula XIII)
wherein:
G1-G2-G3-G4-G5-G6-G7-G8-G9-G10-G11-G12-G13-G14-G15-G16-G17-G18-G19-G20-G21-G22-G23-G24-G25 (Formula VI)
wherein:
(H1)m-(H2)m-(H3)m-(H4)m-(H5)m-(H6)m-(H7)m-(H8)m-(H9)m-(H10)m-(H11)m- (H12)m-(H13)m-(H14)m-(H15)m-(H16)m-(H17)m-(H18)m-(H19)m-(H20)m-(H21)m-(H22)m-(H23)m-(H24)m-(H25)m-(H26)m-(H27)m-(H28)m-(H29)m-(H30)m-(H31)m-(H32)m-(H33)m-(H34)m-(H35)m-(H36)m-H37-H38-H39-H40 (Formula VII)
wherein:
(I1)m-(I2)m-(I3)m-(I4)m-(I5)m-(I6)m-(I7)x-(I8)m-(I9)m-(I10)m-(I11)x- (I12)m-(I13)x-(I14)x-(I15)m-(I16)x-(I17)m-I18-I19-I20-I21-I22-I23 (Formula VIII)
wherein:
(J1)z-(J2)z-(J3)z-(J4)z-(J5)z-(J6)z-(J7)z-(J8)z-(J9)z-(J10)z-(J11)z- (J12)z-(J13)z-(J14)z-(J15)z-(J16)z-(J17)z-(J18)z-(J19)z-(J20)z-(J21)z-J22-J23-J24-J25 (Formula X)
wherein:
(K1)b-(K2)b-(K3)b-(K4)b-(K5)b-(K6)b-(K7)b-(K8)b-(K9)b-(K10)b-(K11)b- (K12)b-(K13)b-(K14)b-(K15)b-(K16)b-(K17)b-(K18)b-(K19)b-(K20)b-(K21)b-(K22)b-(K23)b-(K24)b-(K25)b-(K26)b-(K27)b-(K28)b-(K29)b-(K30)b-(K31)b-(K32)b-(K33)b-(K34)b-(K35)b-(K36)b-(K37)b-(K38)b-(K39)b-(K40)b-(K41)b-(K42)b-(K43)b-(K44)b-(K45)b-(K46)b-(K47)b-(K48)b-(K49)b-(K50)b-(K51)b-(K52)b-(K53)b-(K54)b-(K55)b-(K56)b-(K57)b-(K58)b-(K59)b-(K60)b-(K61)b-(K62)b-(K63)b-(K64)b-(K65)b-(K66)b-(K67)b-(K68)b-(K69)b-(K70)b-(K71)b-(K72)b-(K73)b-(K74)b-(K75)b-(K76)b-(K77)b-(K78)b-(K79)b-(K80)b-(K81)b-(K82)b-(K83)b-(K84)b-(K85)b-(K86)b-(K87)b-(K88)b-K89-K89-K89-K89-K89 (Formula XI)
wherein:
(M1)b-(M2)b-(M3)b-(M4)b-(M5)b-(M6)b-(M7)b-(M8)b-(M9)b-(M10)b-(M11)b- (M12)b-(M13)b-(M14)b-(M15)b-(M16)b-(M17)b-(M18)b-(M19)b-(M20)b-(M21)b-(M22)b-(M23)b-(M24)b-(M25)b-(M26)b-(M27)b-(M28)b-(M29)b-(M30)b-(M31)b-(M32)b-(M33)b-(M34)b-(M35)b-(M36)b-(M37)b-(M38)b-(M39)b-(M40)b-(M41)b-(M42)b-(M43)b-(M44)b-(M45)b-(M46)b-(M47)b-(M48)b-(M49)b-(M50)b-(M51)b-(M52)b-(M53)b-(M54)b-(M55)b-(M56)b-(M57)b-(M58)b-(M59)b-(M60)b-(M61)b-(M62)b-(M63)b-(M64)b-(M65)b-(M66)b-(M67)c-(M68)c-(M69)c-(M70)c (Formula XIV);
wherein:
(N1)b-(N2)b-(N3)b-(N4)b-(N5)b-(N6)b-(N7)b-(N8)b-(N9)b-(N10)b-(N11)b- (N12)b-(N13)b-(N14)b-(N15)b-(N16)b-(N17)b-(N18)b-(N19)b-(N20)b-(N21)b-(N22)b-(N23)b-(N24)b-(N25)b-(N26)b-(N27)b-(N28)b-(N29)b-(N30)b-(N31)b-(N32)b-(N33)b-(N34)b-(N35)b-(N36)b-(N37)b-(N38)b-(N39)b-(N40)b-(N41)b-(N42)b-(N43)b-(N44)b-(N45)b-(N46)b-(N47)b-(N48)b-(N49)b-(N50)b-(N51)b-(N52)b-(N53)b-(N54)b-(N55)b-(N56)b-(N57)b-(N58)b-(N59)b-(N60)b-(N61)b-(N62)b-(N63)b-(N64)b-(N65)b-(N66)b-(N67)c-(N68)c-(N69)c-(N70)c-(N71)c (Formula XV);
wherein:
A1-(A2)w-A3-(A4)x-(A5)y-A6-A7-A8-A9-A10-(A11)z (Formula I)
wherein:
B1-(B2)u-(B3)v-(B4)w-(B5)x-(B6)y-B7-B8-B9-B10-(B11)z (Formula II)
wherein:
C1-(C2)r-(C3)t-(C4)u-[(C5)v-(C6)w]x-(C7)y-(C8)z-C9-C10-C11-[C12-C13]a (Formula III)
wherein:
D1-(D2)q-(D3)r-(D4)t-(D5)u-[(D6)v-(D7)x-(D8)w-(D9)y]z-D10-D11-D12-[D13-D14]a (Formula IV)
wherein:
E1-[(E2)i-(E3)j-(E4)q]r-(E5)t-(E6)u-(E7)v-[(E8)w-(E9)x]y-(E10)z-E11-E12-E13-[E14-E15]a (Formula V)
wherein:
F1-(F2)v-(F3)w-[(F4)x-(F5)y]z-F6-F7-F8-[F9-F10]a (Formula IX)
wherein:
L1-(L2)x-[(L3)a-(L4)a]y-[(L5)a-(L6)a-(L7)a]z-(L8)a-(L9)a-(L10)a-(L11)a-(L12)a (Formula XIII)
wherein:
G1-G2-G3-G4-G5-G6-G7-G8-G9-G10-G11-G12-G13-G14-G15-G16-G17-G18-G19-G20-G21-G22-G23-G24-G25 (Formula VI)
wherein:
(H1)m-(H2)m-(H3)m-(H4)m-(H5)m-(H6)m-(H7)m-(H8)m-(H9)m-(H10)m-(H11)m- (H12)m-(H13)m-(H14)m-(H15)m-(H16)m-(H17)m-(H18)m-(H19)m-(H20)m-(H21)m-(H22)m-(H23)m-(H24)m-(H25)m-(H26)m-(H27)m-(H28)m-(H29)m-(H30)m-(H31)m-(H32)m-(H33)m-(H34)m-(H35)m-(H36)m-H37-H38-H39-H40 (Formula VII)
wherein:
(I1)m-(I2)m-(I3)m-(I4)m-(I5)m-(I6)m-(I7)x-(I8)m-(I9)m-(I10)m-(I11)x- (I12)m-(I13)x-(I14)x-(I15)m-(I16)x-(I17)m-I18-I19-I20-I21-I22-I23 (Formula VIII)
wherein:
(J1)z-(J2)z-(J3)z-(J4)z-(J5)z-(J6)z-(J7)z-(J8)z-(J9)z-(J10)z-(J11)z- (J12)z-(J13)z-(J14)z-(J15)z-(J16)z-(J17)z-(J18)z-(J19)z-(J20)z-(J21)z-J22-J23-J24-J25 (Formula X)
wherein:
(K1)b-(K2)b-(K3)b-(K4)b-(K5)b-(K6)b-(K7)b-(K8)b-(K9)b-(K10)b-(K11)b- (K12)b-(K13)b-(K14)b-(K15)b-(K16)b-(K17)b-(K18)b-(K19)b-(K20)b-(K21)b-(K22)b-(K23)b-(K24)b-(K25)b-(K26)b-(K27)b-(K28)b-(K29)b-(K30)b-(K31)b-(K32)b-(K33)b-(K34)b-(K35)b-(K36)b-(K37)b-(K38)b-(K39)b-(K40)b-(K41)b-(K42)b-(K43)b-(K44)b-(K45)b-(K46)b-(K47)b-(K48)b-(K49)b-(K50)b-(K51)b-(K52)b-(K53)b-(K54)b-(K55)b-(K56)b-(K57)b-(K58)b-(K59)b-(K60)b-(K61)b-(K62)b-(K63)b-(K64)b-(K65)b-(K66)b-(K67)b-(K68)b-(K69)b-(K70)b-(K71)b-(K72)b-(K73)b-(K74)b-(K75)b-(K76)b-(K77)b-(K78)b-(K79)b-(K80)b-(K81)b-(K82)b-(K83)b-(K84)b-(K85)b-(K86)b-(K87)b-(K88)b-K89-K89-K89-K89-K89 (Formula XI)
wherein:
(M1)b-(M2)b-(M3)b-(M4)b-(M5)b-(M6)b-(M7)b-(M8)b-(M9)b-(M10)b-(M11)b- (M12)b-(M13)b-(M14)b-(M15)b-(M16)b-(M17)b-(M18)b-(M19)b-(M20)b-(M21)b-(M22)b-(M23)b-(M24)b-(M25)b-(M26)b-(M27)b-(M28)b-(M29)b-(M30)b-(M31)b-(M32)b-(M33)b-(M34)b-(M35)b-(M36)b-(M37)b-(M38)b-(M39)b-(M40)b-(M41)b-(M42)b-(M43)b-(M44)b-(M45)b-(M46)b-(M47)b-(M48)b-(M49)b-(M50)b-(M51)b-(M52)b-(M53)b-(M54)b-(M55)b-(M56)b-(M57)b-(M58)b-(M59)b-(M60)b-(M61)b-(M62)b-(M63)b-(M64)b-(M65)b-(M66)b-(M67)c-(M68)c-(M69)c-(M70)c (Formula XIV);
wherein:
(N1)b-(N2)b-(N3)b-(N4)b-(N5)b-(N6)b-(N7)b-(N8)b-(N9)b-(N10)b-(N11)b- (N12)b-(N13)b-(N14)b-(N15)b-(N16)b-(N17)b-(N18)b-(N19)b-(N20)b-(N21)b-(N22)b-(N23)b-(N24)b-(N25)b-(N26)b-(N27)b-(N28)b-(N29)b-(N30)b-(N31)b-(N32)b-(N33)b-(N34)b-(N35)b-(N36)b-(N37)b-(N38)b-(N39)b-(N40)b-(N41)b-(N42)b-(N43)b-(N44)b-(N45)b-(N46)b-(N47)b-(N48)b-(N49)b-(N50)b-(N51)b-(N52)b-(N53)b-(N54)b-(N55)b-(N56)b-(N57)b-(N58)b-(N59)b-(N60)b-(N61)b-(N62)b-(N63)b-(N64)b-(N65)b-(N66)b-(N67)c-(N68)c-(N69)c-(N70)c-(N71)c (Formula XV);
wherein:
The functionality and secretion activity of a synthetic signal peptide comprising an amino acid sequence represented by SEQ ID NO. 1 (synKlac-v1) was measured by integrating a nucleic acid encoding synKlac-v1 into a commercially available expression system kit based on K. lactis, substituting the nucleic acid encoding synKlac-v1 for the standard pre-protein signal peptide α-MF. The nucleic acid (DNA) sequence encoding Formula I or SEQ ID NO. 1 is represented by the nucleotide SEQ ID NO. 39, which was obtained from the K. lactis genome.
Secretion of MBP using synthetic signal peptide synKlac-v1 was compared to production utilizing the standard construct comprising the alpha-mating factor from K. lactis (α-MF). To validate the hypothesis that the pro-protein signal peptide and the KR site are not necessary when synKlac-v1 is present, a control recombinant polypeptide that features α-MF without the pro-protein signal peptide and KR site motifs was produced (α-MF (No PPSP).
The secretion efficiency of synKlac-v1, α-MF, and α-MF (No PPSP) was assessed qualitatively and quantitatively by measuring the secretion of MBP in cell-free supernatants obtained from yeast that were grown over several intervals of time, as driven by an inducible galactose promoter.
The results shown in
The western blot data thus obtained were quantified by measuring fluorescent signal intensity generated by the antibodies bound to MBP protein. Data for each recombinant polypeptide variant were plotted over time and cell culture growth. The results, which are shown in
MBP transcript levels for each of the recombinant polypeptide variants were measured to confirm that the detected increases in secretion were not due to increased mRNA transcript production.
Mutations were introduced into synKlac-v1 to identify and design additional synthetic signal peptides capable of directing secretion of a payload protein from yeast. Synthetic signal pre- and pro-protein signal peptides designed according to the disclosed methods were demonstrated to increase secretion of a payload protein in all tested yeast strains, outperforming secretion driven by α-MF, which has been considered the secretion gold standard for the last 30 years.
To validate the secretion efficiency of synKlac-v1 for other payloads, secretion of an anti-TNFα antibody fragment was tested. Secretion was compared between K. lactis strains which secrete anti-TNFα driven by either synKlac-v1 or α-MF. Yeast was grown in inducing medium for 24 hours after which culture supernatant was subjected to ELISA analysis.
Secretion of anti-TNFα antibody fragments from S. boulardii was also investigated. Two synthetic signal peptide variants were tested, Sbou-variant 1 and Sbou-variant 2 (
To expand the methods to other yeast strains routinely used to generate biologics or other bio-commodities, synthetic signal peptides were designed for use and expression in P. pastoris. Four recombinant polypeptide variants, each comprising a synthetic pre-protein signal peptide but lack a pro-protein signal peptide, were cloned into a commercially available expression plasmid for P. pastoris (Pichia Expression Kit—K1710-001, available from Invitrogen®.) A commercially significant version of phytase enzyme from Escherichia coli (Nov9X, ABVista®) was cloned into these plasmids to test the capability of the pre-protein signal peptide to facilitate secretion of phytase enzyme in P. pastoris against to two routinely used signal peptides in Pichia. The constructs of these recombinant polypeptide variants are depicted in
The amount of phytase secreted by P. pastoris strains expressing the signal peptide from S. cerevisiae α-MF (SEQ. ID NO. 2), PHO1 (SEQ ID NO. 30), or the synthetic signal peptide comprising an amino acid sequence represented by SEQ ID NO. 4 (synPichia-v1), SEQ ID NO. 5 (synPichia-v2), SEQ ID NO. 6 (synPichia-v3) or SEQ ID NO. 7 (synPichia-v4) as measured using enzymatic activity assays. Specifically, phytase was indirectly measured through phytase activity, which was estimated by quantifying the amount of free phosphate liberated from a dodecasodium phytate substrate (7.5 mM phytate, 100 mM NaOAc, pH 5.5). Different dilutions of 50 μL P. pastoris culture supernatants (grown for 48 hours in the induction medium (BMMY)) were incubated with 100 μL of the substrate for 1 hour for at 37° C. The reaction was stopped by adding 100 μL of Color Stop reagent (ammonium molybdate, ammonium vanadate and nitric acid) and absorbance at 415 nm was measured. The amount of phytase was quantified using a standard curve generated using purified phytase enzyme from rice. The phytase amounts were then normalized to (divided by) the corresponding CFU of the culture. Normalized phytase yields for each recombinant polypeptide variant in
To test the validity of the approach in designing a superior signal peptide and to expand the approach to the most widely used commercial yeast, S. cerevisiae, several versions of synthetic signal peptides were designed. Synthetic signal peptides contained either a synthetic pre-protein signal peptide or a synthetic pre-protein signal peptide fused with a synthetic pro-protein signal peptide. These synthetic signal peptides were cloned into a plasmid routinely used for expression of insulin in yeast and the secretion of insulin from each was measured and compared to other signal peptides routinely used in the generation of insulin from S. cerevisiae. The performance of recombinant polypeptide variants comprising the synthetic signal peptide comprising an amino acid sequence represented by SEQ ID NO. 9 fused to SEQ ID NO. 20 (synScer-v5) was compared to the performance of α-MF and the Yeast Aspartic Protease 3 (YAP) to secrete insulin.
As shown in
The different versions of an optimized signal peptide with/without synthetic pro-protein signal peptides were also tested for secretion of invertase in S. boulardii, for treatment of sucrose intolerance (e.g., congenital sucrase-isomaltase deficiency, functional gut disorders). Synthetic signal peptides contained either a synthetic pre-protein signal or a synthetic pre-protein signal fused with a synthetic pro-protein signal. Nucleic acids encoding for these synthetic signal peptides were cloned into a plasmid designed for expression of proteins in S. boulardii and the secretion of invertase from each was measured and compared to the native signal peptide present in the endogenous version of SUC2 gene which codes for the native invertase protein in S. boulardii.
As shown in
Additional synthetic peptide variants were also investigated using the same method (Sbou-variant 1-Sbou-variant 12).
The effect of pH on the activity of the invertase enzyme secreted using the synthetic signal peptides was compared that of a pure yeast invertase enzyme obtained from SIGMA.
To reveal the utility of S. boulardii as a delivery agent for invertase in gut, mice were orally administered S. boulardii strains carrying plasmids encoding either invertase with the synthetic signal peptide synScer-v1 (SEQ ID NO. 9 fused to SEQ ID NO. 25) or the native signal peptide by gavage. Mice provided invertase-expressing yeast were then orally administered sucrose. Blood glucose levels were monitored as proxy for invertase activity in mice. The blood glucose levels shown in
S. boulardii yeast were isolated from different tissues of the digestive system of the mice receiving the engineered yeast. Tissues were extracted from each mouse, rinsed in PBS, and then plated at different dilutions on standard growth media with G418 antibiotic. As seen in
The amount of invertase secreted from S. boulardii strains carrying the plasmids encoding invertase with the synthetic signal peptide synScer-v1 were compared with S. boulardii wild type strain. The total amount per CFU was estimated by measuring the invertase activity from S. boulardii periplasmic extracts and dividing that invertase activity by the number of CFUs, which was estimated based upon the corresponding culture's optical density at 600 nm. As seen in
The different versions of an optimized signal peptide with synthetic signal peptides with/without synthetic pro-protein signal peptides were also tested for secretion of IGF-1 in S. boulardii, for treatment of short bowel syndrome. Specific synthetic signal peptides included SEQ ID NO. 9 combined with SEQ ID NO. 20 (synScer-v5), 21 (synScer-v4), or 25 (synScer-v1). Synthetic signal peptides contained either a synthetic pre-protein signal peptide or a synthetic pre-protein signal peptide fused with a synthetic pro-protein signal peptide. Nucleic acids encoding these synthetic signal peptides were cloned into a plasmid designed for expression of insulin-like growth factor 1 (IGF-1) protein in S. boulardii. The secretion of IGF-1 from each was measured using ELISA. Engineered and wild-type S. boulardii strains were grown for 24 hours in standard growth conditions. The level of secreted IGF-1 was quantified by performing ELISA on culture supernatants and then expressed as normalized invertase yields by dividing IGF-1 amount by the number of CFUs, which was estimated based upon the corresponding culture's optical density at 600 nm. As shown in
The different versions of an optimized signal peptide with/without synthetic pro-protein signal peptides were also tested for secretion of lysozyme in S. boulardii, for treatment of small intestinal bacterial overgrowth, pouchitis, C. difficile infection, or any other enteric infection. S. boulardii strains carrying plasmids with nucleic acids encoding lysozyme with the synthetic signal peptides or a signal peptide which is routinely used to secrete protein from yeast such as α-MF were constructed. Specific synthetic signal peptides included SEQ ID NO. 9 combined with SEQ ID NO. 20 (synScer-v5) or 21 (synScer-v4) SEQ ID NO. 9 fused to the S. cerevisiae pro-protein signal peptide α-MF (SEQ ID NO. 2).
Lysozyme activity was estimated from culture supernatants of S. boulardii cultures carrying the different plasmids either encoding for α-MF signal or the synthetic signal peptides and also in swill-type S. boulardii strains without any plasmids as a control. The strain were grown for 72 hours and enzymatic activity of lysozyme was estimated using a commercially available kit. The total amount per CFU was estimated by measuring the lysozyme activity from S. boulardii supernatants, dividing lysozyme activity by the number of CFUs, which was estimated based upon the corresponding culture's optical density at 600 nm and then subtracting the background activity/CFU measured from supernatants of wild-type S. boulardii strains. As seen in
Building on these results, additional synthetic peptide variants were also investigated using the same method (Sbou-variant 1-Sbou-variant 12).
As shown by
Five, healthy, C57BL/6 mice were orally dosed with 109 CFUs of S. boulardii, engineered to express a fluorescent protein (mCherry). The yeast cells were suspended in 300 mL of PBS with no other formulation excipients.
After 1.5, 3, 6, 24, and 48 hours after the oral dose, a mouse was sacrificed and its GI tract was removed imaged with a ThermoFisher, iBright CCD camera. Images are shown in
The resulting images show the yeast survival and fluorescent protein deployment through the upper GI tract for up to 24 hours, with lower GI exposure via packaging into stool in the cecum. The yeast dose is largely depleted by 48 hours, which is consistent with previous literature indicating that S. boulardii is not a GI colonizer. This is an important property with respect to recombinant live biotherapeutics, as regulatory agencies are preferential to non-colonizing/non-engrafting chassis strains.
The engineered yeast, as disclosed herein, retain activity after lyophilization and freeze-drying, which is particularly advantageous given the ease of storage and transport as well as stability of this form. After lyophilization, the engineered yeast, as disclosed herein, exhibits superior activity over wild type yeast across a range of doses in conditions simulating intestinal fluid, against a physiologically representative sucrose challenge (80 mg per mL of intestinal fluid), by at least 3 fold and up to 40 fold when tested using engineered S. boulardii expressing sucrase. With such high activity, the number of CFUs needed to achieve an activity level within range of a commercially available product, e.g., SUCRAID™ (17,000 IU) from a projected 1010 CFU/dose with wild type yeast, which requires at least 1 g of lyophilized product, to 108 CFU/dose with engineered yeast, which may be formulated in a dose of less than or equal to about 250 mg—a critical advantage with respect to minimizing the footprint of a pill for oral consumption, as CSID interventions must be able to be used in children from an early age. This data is presented in
The effect of pH on the activity of the invertase enzyme secreted using the synthetic signal peptides was compared that of a pure yeast invertase enzyme obtained from SIGMA.
Another advantage compared to commercially available yeast products, such as SUCRAID™, is that the sucrase/invertase activity in such commercial yeast is repressed in the presence of glucose. Glucose is a byproduct of the sucrase/invertase activity itself and therefore auto-regulates the enzyme activity invertase/sucrase to lower the activity as glucose byproduct accumulates in the environment. As such, as glucose accumulates, the therapeutic activity of wild-type yeast decreases. In contrast, the engineered yeast disclosed herein utilize an expression system that is additional to the natively expressed enzyme and therefore, sucrase/invertase expression and therefore activity is insensitive to glucose. This was tested by quantifying the activity loss of our engineered S. boulardii expressing sucrase between cultures grown in 2% vs. 0.05% glucose, as compared to wild type S. boulardii. The results are shown in
Twenty-five, healthy, C57BL/6 mice were orally dosed with 109 CFUs of S. boulardii, engineered to express invertase using signaling peptide synScer-v1. The yeast cells were suspended in 300 mL of PBS with no other formulation excipients.
After 1.5, 3, 6, 24, and 48 hours after the oral dose, five mice were sacrificed and its GI tract was removed and the number of CFUs of S. boulardii were quantified by homogenizing GI tissue samples resected at each time point, and plating onto petri dishes with yeast-selective agar. Results are shown in
Nine, healthy, freshly weened, 3 week old C57BL/6 mice were obtained and placed on a sugar-free diet for 1 week, then orally challenged with 2 g/kg of sucrose, and then orally dosed with either 107 CFUs of wild type S. boulardii, S. boulardii engineered to express invertase using signaling peptide synScer-v1, or 300 μL of PBS (three mice per group). The yeast cells were suspended in 300 μL of PBS with no other formulation excipients.
After 15, 30, 60, 90, 120 and 150 minutes after the oral dose, dose activity was measured via quantification of mouse blood glucose levels were recorded using an Accuchek™ glucometer, where an increase in blood glucose is expected as a result of breakdown of the oral sucrose challenge in the GI tract—resulting in an accumulation of glucose byproduct that is absorbed by mouse GI tissue at levels detectable in blood. Results are shown in
The S. boulardii optimized signal peptide with synthetic pro-protein signal peptide was tested for secretion of lactase in S. boulardii, for treatment of lactose intolerance. Synthetic signal peptide contained a synthetic pre-protein signal used with a synthetic pro-protein signal. Nucleic acids encoding for these synthetic signal peptides were cloned into a plasmid designed for expression of proteins in S. boulardii and the secretion of lactase was measured. Wild type S. boulardii cells were used as control.
As seen in
The different versions of an optimized signal peptide with/without synthetic pro-protein signal peptide were also tested for secretion of different versions of anti-TNFα antibody fragments in S. boulardii. anti-TNFα antibodies are used in clinical gold standard therapies for inflammatory diseases, including inflammatory bowel disease in the gut. A monovalent or bivalent form of anti-TNFα antibody fragments delivered by engineered yeast may similarly be used for therapeutic purposes in the gut. Synthetic signal peptides contained either a synthetic pre-protein signal or a synthetic pre-protein signal fused with a synthetic pro-protein signal. Nucleic acids encoding for these synthetic signal peptides were cloned into a plasmid designed for expression of proteins in S. boulardii and the secretion of either a monovalent or a bivalent form of anti-TNFα antibody fragments were analyzed.
As seen in
S. boulardii cells were engineered for stable and reliable expression of invertase by integrating copies of constructs containing the Sbouv2 synthetic signal peptide fused to the invertase into the S. boulardii genome. Multiple loci in the S boulardii genome were used as targets for genomic integration of the invertase expression construct and were engineered using CRISPR-Cas9 mediated gene targeting approach. The target loci used may be genes such as leu2, his3 and ura3 which exist at one location or two copies in diploid S. boulardii cells or a multi-copy locus such as the long terminal repeat (LTR) of the Ty elements in yeast genome which is present at multiple sites within the genome and hence allows for integration of multiple copies. As seen in
The S. boulardii optimized signal peptides with synthetic pro-protein signal peptides were tested for secretion of LCRF in S. boulardii. The LCRF peptide induces release of the peptide hormone cholecystokinin (CCK) or pancreozymin which has important roles in digestion and satiety. Nucleic acids encoding synthetic signal peptide variants (Sbou-variant 1-Sbou-variant 12) were cloned into a plasmid designed for expression of proteins in S. boulardii and the secretion of LCRF was measured. Wild type S. boulardii cells were used as control.
As detailed herein, any pre-protein signal peptide provided can be paired with any pro-protein signal peptide provided. Additionally, any pre or pro-protein signal peptide can be used in the absence of a corresponding pro or pre-protein signal peptide, respectively. Tables 18 and 19 below recite exemplary embodiments of pre-protein signal peptide, pro-protein signal peptide, and payload protein combinations for the various embodiments described herein.
To test the compatibility of engineered yeast as vectors for the prevention of insect infestation in plants, engineered yeast will be generated expressing a recombinant polypeptide comprising insecticides (e.g. Vip1, Vip2, Vip3, Cry proteins, and the like) and one or both of a pre-protein signal peptide as provided for herein and a pro-protein signal peptide as provided for herein. Different combinations will be constructed to provide for the optimal pre and pro protein peptide combination. Plants will be sprayed with yeast expressing the recombinant polypeptide or a control composition and will be allowed to settle. After a pre-determined amount of time, plants from each group will be subject to insect exposure and the ability of the yeast expressing insecticides to prevent insect related damage and infestation will be assessed.
Similarly, the engineered yeast will be assessed for their ability to treat an existing insect infestation. Engineered yeast will be generated expressing a recombinant polypeptide comprising insecticides (e.g. Vip1, Vip2, Vip3, Cry proteins, and the like) and one or both of a pre-protein signal peptide as provided for herein and a pro-protein signal peptide as provided for herein. Different combinations will be constructed to provide for the optimal pre and pro protein peptide combination. Plants will be subject to insect exposure for a pre-determined period of time. Once an infestation is established, plants will be sprayed with either a control composition or a composition comprising the engineered yeast described in this example. The ability of the yeast to clear the existing insect infestation will be assessed.
It should be recognized that illustrated embodiments are only examples of the disclosed product and methods and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/019962 | 3/11/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63159843 | Mar 2021 | US | |
| 63221041 | Jul 2021 | US |
