HUMAN ALPHA-GALACTOSIDASE VARIANTS

Abstract
The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides have been optimized to provide improved stability under both acidic (pH <4.5) and basic (pH >7) conditions. The invention also relates to the use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.
Description
FIELD OF THE INVENTION

The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides have been optimized to provide improved stability under both acidic (pH <4.5) and basic (pH >7) conditions. The invention also relates to the use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.


REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name of “CX7-147W02UC1_ST25.txt”, a creation date of Aug. 4, 2020, and a size of 2,545,695 bytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.


BACKGROUND OF THE INVENTION

Human alpha galactosidase (“GLA”; EC 3.2.1.22) is a lysosomal glycoprotein responsible for hydrolyzing terminal alpha galactosyl moieties from glycolipids and glycoproteins. It works on many substrates present in a range of human tissues. Fabry disease (also referred to as angiokeratoma corporis diffusum, Anderson-Fabry disease, hereditary dystopic lipidosis, alpha-galactosidase A deficiency, GLA deficiency, and ceramide trihexosidase deficiency) is an X-linked inborn error of glycosphingolipid catabolism that results from deficient or absent activity of alpha-galactosidase A. Patients affected with Fabry disease accumulate globotriosylceramide (Gb3) and related glycosphingolipids in the plasma and cellular lysosomes of blood vessels, tissue and organs (See e.g., Nance et al., Arch. Neurol., 63:453-457 [2006]). As the patient ages, the blood vessels become progressively narrowed, due to the accumulation of these lipids, resulting in decreased blood flow and nourishment to the tissues, particularly in the skin, kidneys, heart, brain, and nervous system. Thus, Fabry disease is a systemic disorder that manifests as renal failure, cardiac disease, cerebrovascular disease, small-fiber peripheral neuropathy, and skin lesions, as well as other disorders (See e.g., Schiffmann, Pharm. Ther., 122:65-77 [2009]). Affected patients exhibit symptoms such as painful hands and feet, clusters of small, dark red spots on their skin, the decreased ability to sweat, corneal opacity, gastrointestinal issues, tinnitus, and hearing loss. Potentially life-threatening complications include progressive renal damage, heart attacks, and stroke. This disease affects an estimated 1 in 40,000-60,000 males, but also occurs in females. Indeed, heterozygous women with Fabry disease experience significant life-threatening conditions requiring medical treatment, including nervous system abnormalities, chronic pain, fatigue, high blood pressure, heart disease, kidney failure, and stroke (See e.g., Want et al., Genet. Med., 13:457-484 [2011]). Signs of Fabry disease can start any time from infancy on, with signs usually beginning to show between ages 4 and 8, although some patients exhibit a milder, late-onset disease. Treatment is generally supportive and there is no cure for Fabry disease, thus there remains a need for a safe and effective treatment.


SUMMARY OF THE INVENTION

The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides have been optimized to provide improved stability under both acidic (pH <4.5) and basic (pH >7) conditions. The invention also relates to the use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.


The present invention provides recombinant alpha galactosidase A and/or biologically active recombinant alpha galactosidase A fragment comprising an amino acid sequence comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:5. In some embodiments, the alpha galactosidase A comprises at least one mutation in at least one position as provided in Tables 2.1, 2.2, 2.4, and/or 2.5, wherein the positions are numbered with reference to SEQ ID NO:5. In some embodiments, the alpha galactosidase A comprises at least one mutation in at least one position as provided in Table 2.3, wherein the positions are numbered with reference to SEQ ID NO:10. In some additional embodiments, the recombinant alpha galactosidase A is derived from a human alpha galactosidase A. In some further embodiments, the recombinant alpha galactosidase A comprises the polypeptide sequence of SEQ ID NO:15, 13, 10, or 18. In still some additional embodiments, the recombinant alpha galactosidase A is more thermostable than the alpha galactosidase A of SEQ ID NO:5. In some further embodiments, the recombinant alpha galactosidase A is more stable at pH 7.4 than the alpha galactosidase A of SEQ ID NO:5, while in additional embodiments, the recombinant alpha galactosidase A is more stable at pH 4.3 than the alpha galactosidase A of SEQ ID NO:5. In some embodiments the recombinant alpha galactosidase A is more stable at pH 7.4 and pH 4.3 than the alpha galactosidase A of SEQ ID NO:5. In still some further embodiments, the recombinant alpha galactosidase A is a deimmunized alpha galactosidase A. In some embodiments, the recombinant alpha galactosidase A is a deimmunized alpha galactosidase A provided in Table 7.1. In still some additional embodiments, the recombinant alpha galactosidase A is purified. In some embodiments, the recombinant alpha galactosidase A exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to pH 7.4; iii) increased tolerance to pH 4.3; or iv) reduced immunogenicity; or a combination of any of i), ii), iii), or iv), as compared to a reference sequence. In some embodiments, the reference sequence is SEQ ID NO:5, while in some alternative embodiments, the reference sequence is SEQ ID NO:10.


The present invention also provides recombinant polynucleotide sequences encoding at least one recombinant alpha galactosidase A as provided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In some embodiments, the recombinant polynucleotide sequence is codon-optimized.


The present invention also provides expression vectors comprising the recombinant polynucleotide sequence encoding at least one recombinant alpha galactosidase A as provided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In some embodiments, the recombinant polynucleotide sequence is operably linked to a control sequence. In some additional embodiments, the control sequence is a promoter. In some further embodiments, the promoter is a heterologous promoter. In some embodiments, the expression vector further comprises a signal sequence, as provided herein.


The present invention also provides host cells comprising at least one expression vector as provided herein. In some embodiments, the host cell comprises an expression vector comprising the recombinant polynucleotide sequence encoding at least one recombinant alpha galactosidase A as provided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In some embodiments, the host cell is eukaryotic.


The present invention also provides methods of producing an alpha galactosidase A variant, comprising culturing a host cell provided herein, under conditions that the alpha galactosidase A encoded by the recombinant polynucleotide is produced. In some embodiments, the methods further comprise the step of recovering alpha galactosidase A. In some further embodiments, the methods further comprise the step of purifying the alpha galactosidase A.


The present invention also provides compositions comprising at least one recombinant alpha galactosidase A as provided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1). In some embodiments, the present invention provides pharmaceutical compositions. In some additional embodiments, the present invention provides pharmaceutical compositions for the treatment of Fabry disease, comprising an enzyme composition provided herein. In some embodiments, the pharmaceutical compositions, further comprise a pharmaceutically acceptable carrier and/or excipient. In some additional embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion to a human.


The present invention also provides methods for treating and/or preventing the symptoms of Fabry disease in a subject, comprising providing a subject having Fabry disease, and providing at least one pharmaceutical composition compositions comprising at least one recombinant alpha galactosidase A as provided herein (e.g., Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or Table 7.1), and administering the pharmaceutical composition to the subject. In some embodiments, the symptoms of Fabry disease are ameliorated in the subject. In some additional embodiments, the subject to whom the pharmaceutical composition of the present invention has been administered is able to eat a diet that is less restricted in its fat content than diets required by subjects exhibiting the symptoms of Fabry disease. In some embodiments, the subject is an infant or child, while in some alternative embodiments, the subject is an adult or young adult.


The present invention also provides for the use of the compositions provided herein.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 provides a graph showing the relative activity of different GLA constructs in S. cerevisiae after 2-5 days of culturing.



FIG. 2 provides graphs showing the Absolute (Panel A) and relative (Panel B) activity of GLA variants after incubation at various pHs.



FIG. 3 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants after incubation at various temperatures.



FIG. 4 provides graphs showing the absolute (Panel A&B) and relative (Panel C&D) activity of GLA variants after challenge with buffers that contain increasing amounts of serum.



FIG. 5 provides a graph showing the relative activity of GLA variants expressed in HEK293T cells.



FIG. 6 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various pHs.



FIG. 7 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various temperatures.



FIG. 8 provides graphs showing GLA variant activity remaining after incubation in acidic (Panel A) or basic (Panel B) solutions.



FIG. 9 provides a graph showing the GLA activity recovered in rat serum following administration of GLA variants.





DESCRIPTION OF THE INVENTION

The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides have been optimized to provide improved stability under both acidic (pH <4.5) and basic (pH >7) conditions. The invention also relates to the use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.


In some embodiments, the engineered human alpha-galactosidase polypeptides have been optimized to provide improved stability at various levels. The invention also relates to the use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.


Enzyme replacement therapy for treatment of Fabry disease (e.g., Fabrazyme® agalsidase beta; Genzyme) is available and is considered for eligible individuals. Currently used enzyme replacements therapies are recombinantly expressed forms of the wild-type human GLA. It is known that intravenously administered GLA circulates, becomes endocytosed, and travels to the endosomes/lysosomes of target organs, where it reduces the accumulation of Gb3. These drugs do not completely relieve patient symptoms, as neuropathic pain and transient ischemic attacks continue to occur at reduced rates. In addition, the uptake of GLA by most target organs is poor in comparison to the liver, and the enzyme is unstable at the pH of blood and lysosomes. Thus, issues remain with available treatments. In addition, patients may develop an immune response (IgG and IgE antibodies targeting the administered drug), and suffer severe allergic (anaphylactic) reactions, severe infusion reactions, and even death. The present invention is intended to provide more stable enzymes suitable for treatment of Fabry disease, yet with reduced side effects and improved outcomes, as compared to currently available treatments. Indeed, the present invention is intended to provide recombinant GLA enzymes that have increased stability in blood (pH 7.4), which the enzyme encounters upon injection into the bloodstream. In addition, the enzyme has increased stability at the pH of the lysosome (pH 4.3), the location where the enzyme is active during therapy. Thus, directed evolution of recombinantly expressed human GLA in Saccharomyces cerevisiae, employing high throughput screening of diverse enzyme variant libraries, was used to provide novel GLA variants with desired stability properties. In addition, variant enzymes were screened and their amino acid sequence determined in order to identify novel GLA variants with a predicted reduced immunogenicity. By providing GLA variants with increased pH stability and reduced immunogenicity, the present invention provides compositions and methods suitable for use in patients by increasing patient tolerance of treatment and providing flexibility in dosing and formulation for improved patient outcomes.


Abbreviations and Definitions

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, biochemistry, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well-known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.


Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the application as a whole. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.


Also, as used herein, the singular “a”, “an,” and “the” include the plural references, unless the context clearly indicates otherwise.


Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.


The term “about” means an acceptable error for a particular value. In some instances “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value.


Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.


Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.


As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).


“EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.


“ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.


“NCBI” refers to National Center for Biological Information and the sequence databases provided therein.


“Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).


“Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.


The term “engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refers to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.


As used herein, “wild-type” and “naturally-occurring” refer to the form found in nature. For example a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.


“Deimmunized” as used herein, refers to the manipulation of a protein sequence to create a variant that is predicted to be not as immunogenic as the wild-type or reference protein. In some embodiments, the predicted deimmunization is complete, in that the variant protein is predicted to not stimulate an immune response in patients to whom the variant protein is administered. This response can be measured by various methods including but not limited to, the presence or abundance of anti-drug antibodies, the presence or abundance of neutralizing antibodies, the presence of an anaphylactic response, peptide presentation on major histocompatibility complex-II (MHC-II) proteins, or the prevalence or intensity of cytokine release upon administration of the protein. In some embodiments, the variant protein is less immunogenic than the wild-type or reference protein. In some embodiments, deimmunization involves modifications to subsequences of proteins (e.g., epitopes) that are recognized by human leukocyte antigen (HLA) receptors. In some embodiments, these epitopes are removed by changing their amino acid sequences to produce a deimmunized variant protein in which such subsequences are no longer recognized by the HLA receptors. In some other embodiments, these epitopes retain binding affinity to HLA receptors, but are not presented. In some embodiments, the deimmunized protein shows lower levels of response in biochemical and cell-biological predictors of human immunological responses including dendritic-cell T-cell activation assays, or (HLA) peptide binding assays. In some embodiments, these epitopes are removed by changing their amino acid sequence to produce a deimmunized variant protein in which the epitopes are no longer recognized by T-cell receptors. In still other embodiments the deimmunized protein induces anergy in its corresponding T-cells, activates T regulatory cells, or results in clonal deletion of recognizing B-cells.


“Coding sequence” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.


The term “percent (%) sequence identity” is used herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482 [1981]), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443 [1970), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See, Altschul et al., J. Mol. Biol., 215: 403-410 [1990]; and Altschul et al., 1977, Nucleic Acids Res., 3389-3402 [1977], respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (See, Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989]). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.


“Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.


“Corresponding to”, “reference to” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered GLA, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.


“Amino acid difference” or “residue difference” refers to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X93 as compared to SEQ ID NO:2” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has a serine at position 93, then a “residue difference at position X93 as compared to SEQ ID NO:2” an amino acid substitution of any residue other than serine at the position of the polypeptide corresponding to position 93 of SEQ ID NO:2. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances (e.g., in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and 6.1), the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “/” (e.g., X307H/X307P or X307H/P). In some embodiments, the enzyme variants comprise more than one substitution. These substitutions are separated by a slash for ease in reading (e.g., C143A/K206A). The present application includes engineered polypeptide sequences comprising one or more amino acid differences that include either/or both conservative and non-conservative amino acid substitutions.


“Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.


“Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.


“Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and/or retaining the improved properties of an engineered enzyme. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.


“Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.


A “functional fragment” or a “biologically active fragment” used interchangeably herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered GLA of the present invention) and that retains substantially all of the activity of the full-length polypeptide.


“Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant GLA polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant GLA polypeptides can be an isolated polypeptide.


“Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure GLA composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant GLA polypeptides are substantially pure polypeptide compositions.


“Improved enzyme property” refers to an engineered GLA polypeptide that exhibits an improvement in any enzyme property as compared to a reference GLA polypeptide and/or as a wild-type GLA polypeptide or another engineered GLA polypeptide. Improved properties include but are not limited to such properties as increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic or basic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, improved post-translational modification (e.g., glycosylation), and altered temperature profile.


“Increased enzymatic activity” or “enhanced catalytic activity” refers to an improved property of the engineered GLA polypeptides, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of GLA) as compared to the reference GLA enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vm, or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.1 fold the enzymatic activity of the corresponding wild-type enzyme, to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more enzymatic activity than the naturally occurring GLA or another engineered GLA from which the GLA polypeptides were derived.


In some embodiments, the engineered GLA polypeptides have a kcat of at least 0.1/sec, at least 0.5/sec, at least 1.0/sec, at least 5.0/sec, at least 10.0/sec and in some preferred embodiments greater than 10.0/sec. In some embodiments, the Km is in the range of about 1 μM to about 5 mM; in the range of about 5 μM to about 2 mM; in the range of about 10 μM to about 2 mM; or in the range of about 10 μM to about 1 mM. In some specific embodiments, the engineered GLA enzyme exhibits improved enzymatic activity after exposure to certain conditions in the range of 1.5 to 10 fold, 1.5 to 25 fold, 1.5 to 50 fold, 1.5 to 100 fold or greater than that of a reference GLA enzyme (e.g., a wild-type GLA or any other reference GLA). GLA activity can be measured by any suitable method known in the art (e.g., standard assays, such as monitoring changes in spectrophotometric properties of reactants or products). In some embodiments, the amount of products produced can be measured by High-Performance Liquid Chromatography (HPLC) separation combined with UV absorbance or fluorescent detection directly or following o-phthaldialdehyde (OPA) derivatization. Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.


The term “improved tolerance to acidic pH” means that a recombinant GLA according to the invention will have increased stability (higher retained activity at about pH 4.8 after exposure to acidic pH for a specified period of time (1 hour, up to 24 hours)) as compared to a reference GLA or another enzyme.


“Physiological pH” as used herein means the pH range generally found in a subject's (e.g., human) blood.


The term “basic pH” (e.g., used with reference to improved stability to basic pH conditions or increased tolerance to basic pH) means a pH range of about 7 to 11.


The term “acidic pH” (e.g., used with reference to improved stability to acidic pH conditions or increased tolerance to acidic pH) means a pH range of about 1.5 to 4.5.


“Conversion” refers to the enzymatic conversion (or biotransformation) of a substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of a GLA polypeptide can be expressed as “percent conversion” of the substrate to the product in a specific period of time.


“Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. “High stringency hybridization” refers generally to conditions that are about 10° C. or less from the thermal melting temperature Tm as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v) SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.


“Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the GLA enzymes may be codon optimized for optimal production from the host organism selected for expression.


“Control sequence” refers herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.


“Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest.


“Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.


“Suitable reaction conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a GLA polypeptide of the present application is capable of converting a substrate to the desired product compound, Exemplary “suitable reaction conditions” are provided in the present application and illustrated by the Examples. “Loading”, such as in “compound loading” or “enzyme loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction. “Substrate” in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the GLA polypeptide. “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the GLA polypeptide on a substrate.


As used herein the term “culturing” refers to the growing of a population of microbial cells under any suitable conditions (e.g., using a liquid, gel or solid medium).


Recombinant polypeptides can be produced using any suitable methods known the art. Genes encoding the wild-type polypeptide of interest can be cloned in vectors, such as plasmids, and expressed in desired hosts, such as E. coli, S. cerevisiae, etc. Variants of recombinant polypeptides can be generated by various methods known in the art. Indeed, there is a wide variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available to make specific substitutions at defined amino acids (site-directed), specific or random mutations in a localized region of the gene (regio-specific), or random mutagenesis over the entire gene (e.g., saturation mutagenesis). Numerous suitable methods are known to those in the art to generate enzyme variants, including but not limited to site-directed mutagenesis of single-stranded DNA or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein engineering are provided in the following patents: U.S. Pat. Nos. 6,117,679; 6,420,175; 6,376,246; 6,586,182; 7,747,391; 7,747,393; 7,783,428; and 8,383,346. After the variants are produced, they can be screened for any desired property (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermal stability, and/or acidic pH stability, etc.). In some embodiments, “recombinant GLA polypeptides” (also referred to herein as “engineered GLA polypeptides,” “variant GLA enzymes,” and “GLA variants”) find use.


As used herein, a “vector” is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polypeptide encoded in the DNA sequence. In some embodiments, an “expression vector” has a promoter sequence operably linked to the DNA sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.


As used herein, the term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.


As used herein, the term “produces” refers to the production of proteins and/or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.


As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is “heterologous” to another sequence with which it is operably linked if the two sequences are not associated in nature.


As used herein, the terms “host cell” and “host strain” refer to suitable hosts for expression vectors comprising DNA provided herein (e.g., the polynucleotides encoding the GLA variants). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.


The term “analogue” means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, analogues mean polypeptides that contain one or more non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.


The term “therapeutic” refers to a compound administered to a subject who shows signs or symptoms of pathology having beneficial or desirable medical effects.


The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., human) comprising a pharmaceutically effective amount of an engineered GLA polypeptide encompassed by the invention and an acceptable carrier.


The term “effective amount” means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount by using routine experimentation.


The terms “isolated” and “purified” are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term “purified” does not require absolute purity, rather it is intended as a relative definition.


The term “subject” encompasses mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagamorphs). It is intended that the term encompass females as well as males.


As used herein, the term “patient” means any subject that is being assessed for, treated for, or is experiencing disease.


The term “infant” refers to a child in the period of the first month after birth to approximately one (1) year of age. As used herein, the term “newborn” refers to child in the period from birth to the 28th day of life. The term “premature infant” refers to an infant born after the twentieth completed week of gestation, yet before full term, generally weighing ˜500 to ˜2499 grams at birth. A “very low birth weight infant” is an infant weighing less than 1500 g at birth.


As used herein, the term “child” refers to a person who has not attained the legal age for consent to treatment or research procedures. In some embodiments, the term refers to a person between the time of birth and adolescence.


As used herein, the term “adult” refers to a person who has attained legal age for the relevant jurisdiction (e.g., 18 years of age in the United States). In some embodiments, the term refers to any fully grown, mature organism. In some embodiments, the term “young adult” refers to a person less than 18 years of age, but who has reached sexual maturity.


As used herein, “composition” and “formulation” encompass products comprising at least one engineered GLA of the present invention, intended for any suitable use (e.g., pharmaceutical compositions, dietary/nutritional supplements, feed, etc.).


The terms “administration” and “administering” a composition mean providing a composition of the present invention to a subject (e.g., to a person suffering from the effects of Fabry disease).


The term “carrier” when used in reference to a pharmaceutical composition means any of the standard pharmaceutical carrier, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.


The term “pharmaceutically acceptable” means a material that can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the components in which it is contained and that possesses the desired biological activity.


As used herein, the term “excipient” refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API; e.g., the engineered GLA polypeptides of the present invention). Excipients are typically included for formulation and/or administration purposes.


The term “therapeutically effective amount” when used in reference to symptoms of disease/condition refers to the amount and/or concentration of a compound (e.g., engineered GLA polypeptides) that ameliorates, attenuates, or eliminates one or more symptom of a disease/condition or prevents or delays the onset of symptom(s).


The term “therapeutically effective amount” when used in reference to a disease/condition refers to the amount and/or concentration of a composition (e.g., engineered GLA polypeptides) that ameliorates, attenuates, or eliminates the disease/condition. In some embodiments, the term is use in reference to the amount of a composition that elicits the biological (e.g., medical) response by a tissue, system, or animal subject that is sought by the researcher, physician, veterinarian, or other clinician.


It is intended that the terms “treating,” “treat” and “treatment” encompass preventative (e.g., prophylactic), as well as palliative treatment.


Engineered GLA Expression and Activity:

Two strategies for secreted GLA expression were utilized, using the yeast MFα signal peptide (MF-SP) or a longer leader sequence of 83 amino acids (MF-leader) to drive secretion of a yeast codon-optimized mature human GLA. Clones were expressed from a pYT-72 vector in S. cerevisiae strain INVSc1. Both approaches provided supernatants with measurable activity on the fluorogenic substrate 4-methylumbelliferyl α-D-galactopyranoside (4-MuGal). However, the construct with the yeast MFα signal peptide provided 3-fold higher activities and was used as the starting sequence for directed evolution.


To identify mutational diversity, a 13-position conserved “homolog” combinatorial library and a 192-position site saturation mutagenesis library were constructed. Equivalent volumes of supernatant were screened in an unchallenged condition (no incubation, pH 4.8) or following a one-hour incubation in a low pH (3.9-4.2) or high pH (7.1-8.2) environment. GLA variants with increased activity due to increased GLA expression or GLA specific activity were identified based on their fold improvement over the parent GLA. GLA variants with increased stability were identified by dividing the fold-improvement observed under challenged conditions by the fold-improvement observed under unchallenged conditions. This approach reduces the bias towards selecting variants based on increased expression but without changes in specific activity at pH extremes. Composite activity scores (the product of fold-improvements for all three conditions) and stability (the product of stability scores) were used to rank mutations in improved variants for inclusion in subsequent GLA libraries.


Engineered GLA:

In some embodiments the engineered GLA which exhibits an improved property has at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at about 100% amino acid sequence identity with SEQ ID NO:5, and an amino acid residue difference as compared to SEQ ID NO:5, at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or more amino acid positions compared to SEQ ID NO:5, or a sequence having at least 85%, at least 88%, 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%, at least 99% or greater amino acid sequence identity with SEQ ID NO:5). In some embodiment the residue difference as compared to SEQ ID NO:5, at one or more positions will include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the engineered GLA polypeptide is a polypeptide listed in Table 2.1, 2.2, 2.4, 2.5, or Table 7.1.


In some embodiments the engineered GLA which exhibits an improved property has at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at about 100% amino acid sequence identity with SEQ ID NO:10, and an amino acid residue difference as compared to SEQ ID NO:10, at one or more amino acid positions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or more amino acid positions compared to SEQ ID NO:10, or a sequence having at least 85%, at least 88%, 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%, at least 99% or greater amino acid sequence identity with SEQ ID NO:10). In some embodiment the residue difference as compared to SEQ ID NO:10, at one or more positions will include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the engineered GLA polypeptide is a polypeptide listed in Table 2.3.


In some embodiments the engineered GLA which exhibits an improved property has at least 85%, at least 88%, 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% amino acid sequence identity with SEQ ID NO:5. In some embodiments the engineered GLA which exhibits an improved property has at least 85%, at least 88%, 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% amino acid sequence identity with SEQ ID NO:10.


In some embodiments, the engineered GLA polypeptide is selected from SEQ ID NOS:15, 13, 10, and 18.


In some embodiments, the engineered GLA polypeptide comprises a functional fragment of an engineered GLA polypeptide encompassed by the invention. Functional fragments have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the engineered GLA polypeptide from which is was derived (i.e., the parent engineered GLA). A functional fragment comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even 99% of the parent sequence of the engineered GLA. In some embodiments the functional fragment is truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids.


Polynucleotides Encoding Engineered Polypeptides, Expression Vectors and Host Cells:

The present invention provides polynucleotides encoding the engineered GLA polypeptides described herein. In some embodiments, the polynucleotides are operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the engineered GLA polypeptides can be introduced into appropriate host cells to express the corresponding GLA polypeptide.


As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode the engineered GLA polypeptide. Thus, having knowledge of a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each and every possible variation of polynucleotides that could be made encoding the polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the variants provided in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and 6.1.


In various embodiments, the codons are preferably selected to fit the host cell in which the protein is being produced. For example, preferred codons used in bacteria are used for expression in bacteria. Consequently, codon optimized polynucleotides encoding the engineered GLA polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full length coding region.


In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having GLA activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOS:5, and/or 10, or the amino acid sequence of any variant as disclosed in Tables 2.1, 2.2, 2.3, 2.4, 2.5, or 6.1, and one or more residue differences as compared to the reference polypeptide of SEQ ID NOS:5, and/or 10, or the amino acid sequence of any variant as disclosed in Tables 2.1, 2.2, 2.3, 2.4, 2.5, or 6.1, (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). In some embodiments, the reference sequence is selected from SEQ ID NO:5 and/or 10. In some embodiments, the polynucleotide encodes an engineered polypeptide having GLA activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO:5, and one or more residue differences as compared to SEQ ID NO:5, at residue positions selected from those provided in Tables 2.1, 2.2, 2.4, 2.5, or 6.1, when optimally aligned with the polypeptide of SEQ ID NO:5.


In some embodiments, the polynucleotide encodes an engineered polypeptide having GLA activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO:10, and one or more residue differences as compared to SEQ ID NO:10, at residue positions selected from those provided in Tables 2.3, when optimally aligned with the polypeptide of SEQ ID NO:10.


In some embodiments, the polynucleotide encoding the engineered GLA polypeptides comprises a polynucleotide sequence selected from a polynucleotide sequence encoding SEQ ID NOS:10, 13, 15, 18, 21, and 24. In some embodiments, the polynucleotide encoding an engineered GLA polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 93%, 95%, 96%, 97%, 98%, 99% nucleotide residue identity to SEQ ID NOS: 8, 9, 11, 12, 14, 16, 17, 19, 20, 22, and/or 23. In some embodiments, the polynucleotide encoding the engineered GLA polypeptides comprises a polynucleotide sequence selected from SEQ ID NOS:8, 9, 11, 12, 14, 16, 17, 19, 20, 22, and 23.


In some embodiments, the polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from SEQ ID NOS: 8, 9, 11, 12, 14, 16, 17, 19, 20, 22, and 23, or a complement thereof, or a polynucleotide sequence encoding any of the variant GLA polypeptides provided herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a GLA polypeptide comprising an amino acid sequence that has one or more residue differences as compared to SEQ ID NO:5 and/or 10, at residue positions selected from any positions as set forth in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and/or 6.1.


In some embodiments, an isolated polynucleotide encoding any of the engineered GLA polypeptides provided herein is manipulated in a variety of ways to provide for expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptides are provided as expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.


In some embodiments, the control sequences include among other sequences, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. As known in the art, suitable promoters can be selected based on the host cells used. Exemplary promoters for filamentous fungal host cells, include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992]). Exemplary promoters for use in mammalian cells include, but are not limited to those from cytomegalovirus (CMV), Simian vacuolating virus 40 (SV40), from Homo sapiens phosphorglycerate kinase, beta actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or from Gallus gallus' β-actin.


In some embodiments, the control sequence is a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice finds use in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra). Exemplary terminators for mammalian cells include, but are not limited to those from cytomegalovirus (CMV), Simian vacuolating virus 40 (SV40), or from Homo sapiens growth hormone.


In some embodiments, the control sequence is a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to those obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).


The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (See e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990 [1995]).


In some embodiments, the control sequence is a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered GLA polypeptides provided herein. Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include but are not limited to those from the genes for immunoglobulin gamma (IgG).


In some embodiments, the control sequence is a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen,” in some cases). A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.


In another aspect, the present invention also provides a recombinant expression vector comprising a polynucleotide encoding an engineered GLA polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. in some embodiments, the various nucleic acid and control sequences described above are joined together to produce a recombinant expression vector which includes one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the variant GLA polypeptide at such sites. Alternatively, the polynucleotide sequence(s) of the present invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.


The recombinant expression vector may be any vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the variant GLA polynucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.


In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.


In some embodiments, the expression vector preferably contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. In another aspect, the present invention provides a host cell comprising a polynucleotide encoding at least one engineered GLA polypeptide of the present application, the polynucleotide being operatively linked to one or more control sequences for expression of the engineered GLA enzyme(s) in the host cell. Host cells for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include but are not limited to, fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [e.g., ATCC Accession No. 201178]); insect cells (e.g., Drosophila S2 and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, COS, and BHK), and human cells (e.g., HEK293T, human fibroblast, THP-1, Jurkat and Bowes melanoma cell lines).


Accordingly, in another aspect, the present invention provides methods for producing the engineered GLA polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered GLA polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the methods further comprise the steps of isolating and/or purifying the GLA polypeptides, as described herein.


Appropriate culture media and growth conditions for the above-described host cells are well known in the art. Polynucleotides for expression of the GLA polypeptides may be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.


The engineered GLA with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered GLA polypeptide to mutagenesis and/or directed evolution methods known in the art, and as described herein. An exemplary directed evolution technique is mutagenesis and/or DNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746). Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (See e.g., Zhao et al., Nat. Biotechnol., 16:258-261 [1998]), mutagenic PCR (See e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140 [1994]), and cassette mutagenesis (See e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529 [1996]).


For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,489,146, 6,506,602, 6,506,603, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,613,514, 6,653,072, 6,716,631, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,288,375, 7,421,347, 7,430,477, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,873,499, 7,904,249, 7,957,912, 8,383,346, 8,504,498, 8,849,575, 8,876,066, 8,768,871, and all related non-US counterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; US Pat. Appln. Publn. Nos. 2008/0220990, US 2009/0312196, US2014/0005057, US2014/0214391, US2014/0221216; US2015/0050658, US2015/0133307, US2015/0134315 and all related non-US counterparts; WO 95/22625, WO 97/0078, WO 97/35966, WO 98/27230, WO 00/42651, WO 01/75767, and WO 2009/152336; all of which are incorporated herein by reference).


In some embodiments, the enzyme variants obtained following mutagenesis treatment are screened by subjecting the enzyme variants to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatments or other assay conditions. DNA containing the polynucleotide encoding the GLA polypeptide is then isolated from the host cell, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a different or the same host cell. Measuring enzyme activity from the expression libraries can be performed using any suitable method known in the art (e.g., standard biochemistry techniques, such as HPLC analysis).


For engineered polypeptides of known sequence, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al., Tetra. Lett., 22:1859-69 [1981]; and Matthes et al., EMBO J., 3:801-05 [1984]), as it is typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer), purified, annealed, ligated and cloned in appropriate vectors.


Accordingly, in some embodiments, a method for preparing the engineered GLA polypeptide can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant provided in Table 2.1, 2.2, 2.3, 2.4, 2.5, and/or 6.1, as well as SEQ ID NOS:10, 13, 15, 18, 21, and/or 24, and (b) expressing the GLA polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and/or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions.


The expressed engineered GLA polypeptide can be assessed for any desired improved property (e.g., activity, selectivity, stability, acid tolerance, protease sensitivity, etc.), using any suitable assay known in the art, including but not limited to the assays and conditions described herein.


In some embodiments, any of the engineered GLA polypeptides expressed in a host cell are recovered from the cells and/or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography.


Chromatographic techniques for isolation of the GLA polypeptides include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the improved variant GLA enzymes. In some embodiments utilizing affinity chromatography purification, any antibody which specifically binds the variant GLA polypeptide finds use. In some embodiments utilizing affinity chromatography purification, proteins that bind to the glycans covalently attached to GLA find use. In still other embodiments utilizing affinity-chromatography purifications, any small molecule that binds to the GLA active site finds use. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., are immunized by injection with a GLA polypeptide (e.g., a GLA variant), or a fragment thereof. in some embodiments, the GLA polypeptide or fragment is attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group.


In some embodiments, the engineered GLA polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., S. cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens (e.g., HEK293T), or Cricetulus griseus (e.g., CHO)) comprising a polynucleotide sequence encoding an engineered GLA polypeptide as described herein under conditions conducive to the production of the engineered GLA polypeptide and recovering the engineered GLA polypeptide from the cells and/or culture medium.


In some embodiments, the invention encompasses a method of producing an engineered GLA polypeptide comprising culturing a recombinant eukaryotic cell comprising a polynucleotide sequence encoding an engineered GLA polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to reference sequences SEQ ID NOS:5 and/or 10, and one or more amino acid residue differences as compared to SEQ ID NO:5 and/or 10, selected from those provided in Tables 2.1, 2.2, 2.4, 2.5, and/or 6.1, and/or combinations thereof when optimally aligned with the amino acid sequence of SEQ ID NO:5 and/or 10, under suitable culture conditions to allow the production of the engineered GLA polypeptide and optionally recovering the engineered GLA polypeptide from the culture and/or cultured bacterial cells.


In some embodiments, once the engineered GLA polypeptides are recovered from the recombinant host cells or cell culture medium, they are further purified by any suitable method(s) known in the art. In some additional embodiments, the purified GLA polypeptides are combined with other ingredients and compounds to provide compositions and formulations comprising the engineered GLA polypeptide as appropriate for different applications and uses (e.g., pharmaceutical compositions). In some additional embodiments, the purified GLA polypeptides, or the formulated GLA polypeptides are lyophilized.


Compositions:

The present invention provides various compositions and formats, including but not limited to those described below. In some embodiments, the present invention provides engineered GLA polypeptides suitable for use in pharmaceutical and other compositions, such as dietary/nutritional supplements.


Depending on the mode of administration, these compositions comprising a therapeutically effective amount of an engineered GLA according to the invention are in the form of a solid, semi-solid, or liquid. In some embodiments, the compositions include other pharmaceutically acceptable components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. Details on techniques for formulation and administration are well known in the art and described in the literature.


In some embodiments, the engineered GLA polypeptides are formulated for use in pharmaceutical compositions. Any suitable format for use in delivering the engineered GLA polypeptides find use in the present invention, including but not limited to pills, tablets, gel tabs, capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks, solutions, suspensions (including but not limited to oil-based suspensions, oil-in water emulsions, etc.), slurries, syrups, controlled release formulations, suppositories, etc. In some embodiments, the engineered GLA polypeptides are provided in a format suitable for injection or infusion (i.e., in an injectable formulation). In some embodiments, the engineered GLA polypeptides are provided in biocompatible matrices such as sol-gels, including silica-based (e.g., oxysilane) sol-gels. In some embodiments, the engineered GLA polypeptides are encapsulated. In some alternative embodiments, the engineered GLA polypeptides are encapsulated in nanostructures (e.g., nanotubes, nanotubules, nanocapsules, or microcapsules, microspheres, liposomes, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery formulation and/or means of delivery. It is intended that the engineered GLA polypeptides be administered by any suitable means known in the art, including but not limited to parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, via implants, etc.


In some embodiments, the engineered GLA polypeptides are chemically modified by glycosylation, chemical crosslinking reagents, pegylation (i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.) or other compounds (See e.g., Ikeda, Amino Acids 29:283-287 [2005]; U.S. Pat. Nos. 7,531,341, 7,534,595, 7,560,263, and 7,53,653; US Pat. Appln. Publ. Nos. 2013/0039898, 2012/0177722, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery method and/or mechanism.


In some additional embodiments, the engineered GLA polypeptides are provided in formulations comprising matrix-stabilized enzyme crystals. In some embodiments, the formulation comprises a cross-linked crystalline engineered GLA enzyme and a polymer with a reactive moiety that adheres to the enzyme crystals. The present invention also provides engineered GLA polypeptides in polymers.


In some embodiments, compositions comprising the engineered GLA polypeptides of the present invention include one or more commonly used carrier compounds, including but not limited to sugars (e.g., lactose, sucrose, mannitol, and/or sorbitol), starches (e.g., corn, wheat, rice, potato, or other plant starch), cellulose (e.g., methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxy-methylcellulose), gums (e.g., arabic, tragacanth, guar, etc.), and/or proteins (e.g., gelatin, collagen, etc.).


In some embodiments, the present invention provides engineered GLA polypeptides suitable for use in decreasing the concentration of glycolipids in fluids such as blood, cerebrospinal fluid, etc. The dosage of engineered GLA polypeptide(s) administered depends upon the condition or disease, the general condition of the subject, and other factors known to those in the art. In some embodiments, the compositions are intended for single or multiple administrations. In some embodiments, it is contemplated that the concentration of engineered GLA polypeptide(s) in the composition(s) administered to a human with Fabry disease is sufficient to effectively treat, and/or ameliorate disease (e.g., Fabry disease). In some embodiments, the engineered GLA polypeptides are administered in combination with other pharmaceutical and/or dietary compositions.


EXPERIMENTAL

The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.


In the experimental disclosure below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and 1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); ° C. (degrees Centigrade); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, Conn.); HPLC (high pressure liquid chromatography); MWCO (molecular weight cut-off); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); PES (polyethersulfone); CFSE (carboxyfluorescein succinimidyl ester); IPTG (isopropyl β-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH (nicotinamide adenine dinucleotide phosphate); GIDH (glutamate dehydrogenase); FIOPC (fold improvements over positive control); PBMC (peripheral blood mononuclear cells); LB (Luria broth); MeOH (methanol); Athens Research (Athens Research Technology, Athens, Ga.); ProSpec (ProSpec Tany Technogene, East Brunswick, N.J.); Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Ram Scientific (Ram Scientific, Inc., Yonkers, N.Y.); Pall Corp. (Pall, Corp., Pt. Washington, N.Y.); Millipore (Millipore, Corp., Billerica Mass.); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, Mich.); Molecular Devices (Molecular Devices, LLC, Sunnyvale, Calif.); Kuhner (Adolf Kuhner, AG, Basel, Switzerland); Axygen (Axygen, Inc., Union City, Calif.); Toronto Research Chemicals (Toronto Research Chemicals Inc., Toronto, Ontario, Canada); Cambridge Isotope Laboratories, (Cambridge Isotope Laboratories, Inc., Tewksbury, Mass.); Applied Biosystems (Applied Biosystems, part of Life Technologies, Corp., Grand Island, N.Y.), Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); Thermo Scientific (part of Thermo Fisher Scientific, Waltham, Mass.); Corning (Corning, Inc., Palo Alto, Calif.); Megazyme (Megazyme International, Wicklow, Ireland); Enzo (Enzo Life Sciences, Inc., Farmingdale, N.Y.); GE Healthcare (GE Healthcare Bio-Sciences, Piscataway, N.J.); Pierce (Pierce Biotechnology (now part of Thermo Fisher Scientific), Rockford, Ill.); LI-COR (LI-COR Biotechnology, Lincoln, Nebr.); Amicus (Amicus Therapeutics, Cranbury, N.J.); Phenomenex (Phenomenex, Inc., Torrance, Calif.); Optimal (Optimal Biotech Group, Belmont, Calif.); and Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.).


The following polynucleotide and polypeptide sequences find use in the present invention. In some cases (as shown below), the polynucleotide sequence is followed by the encoded polypeptide.










Polynucleotide sequence of full length human GLA cDNA (SEQ



ID NO. 1):


(SEQ ID NO: 1)



ATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTGGCC






CTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCT





ACCATGGGCTGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCC





AGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAG





GCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATGGCTCCCCAAA





GAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGC





TAGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAAT





AAAACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTT





GCTGACTGGGGAGTAGATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAAT





TTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGACTGGCAGAAGCATTGTG





TACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAAATC





CGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATA





AAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCA





GGGGGTTGGAATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAG





CAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACC





TCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGTAATTGCCATCA





ATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTG





TGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATT





GGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAAT





CCTGCCTGCTTCATCACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGG





ACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTTTTGCTTCAGCTAGAAAAT





ACAATGCAGATGTCATTAAAAGACTTACTTTAG





Polypeptide sequence of full length human GLA:


(SEQ ID NO: 2)



MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEP






DSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLA





NYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLAD





GYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILD





WTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHIS





PQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSY





TIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKD





LL





Polynucleotide sequence of mature yeast codon-optimized (yCDS)


human GLA:


(SEQ ID NO: 3)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG





GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA





CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA





TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC





TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC





CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT





AAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACT





GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polynucleotide sequence of mature human GLA (native hCDS):


(SEQ ID NO: 4)



CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT






GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG





AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC





ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC





TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA





GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG





ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA





TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC





CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC





CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC





TGACATTGATGATTCCTGGAAAAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA





GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA





TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA





TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT





CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC





AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG





GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT





GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT





GTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCC





CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT





T





Polypeptide sequence of mature Human GLA (SEQ ID NO.5):


(SEQ ID NO: 5)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG





LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG





DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF





YEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of pCK110900i E. coli expression vector:


(SEQ ID NO: 6)



TCGAGTTAATTAAGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGC






ACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATA





ACAATTTCACACAGGAAACGGCTATGACCATGATTACGGATTCACTGGCCGTCGTTTTAC





AATCTAGAGGCCAGCCTGGCCATAAGGAGATATACATATGAGTATTCAACATTTCCGTGT





CGCCCTTATTCCCTTTTCTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG





GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA





TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAGCGTTTTCCAATGATGAG





CACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCA





ACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA





AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGA





GTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACC





GTTTTTTTGCACACCATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTG





AATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAAC





GTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGA





CTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG





GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACT





GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA





CTATGGATGAACGTAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGG





GGCCAAACTGGCCACCATCACCATCACCATTAGGGAAGAGCAGATGGGCAAGCTTGACC





TGTGAAGTGAAAAATGGCGCACATTGTGCGACATTTTTTTTTGAATTCTACGTAAAAAGC





CGCCGATACATCGGCTGCTTTTTTTTTGATAGAGGTTCAAACTTGTGGTATAATGAAATA





AGATCACTCCGGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAA





ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGA





ACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGA





TATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTAT





TCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAGTTCCGTATGGCAATGAAAGACGG





TGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGA





AACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATA





TTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGA





GAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTG





GCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGC





GACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCAT





GTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTA





ACTGCAGGAGCTCAAACAGCAGCCTGTATTCAGGCTGCTTTTTTCGTTTTGGTCTGCGCGT





AATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTG





AGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTG





TCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTAC





CAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGT





TACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTG





GAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATA





ACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCC





GCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGA





GCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCG





CGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGT





TCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGA





GGAAGCGGAATATATCCTGTATCACATATTCTGCTGACGCACCGGTGCAGCCTTTTTTCT





CCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGAACCACCGCTGGTAGCGGTGG





TTTTTTTAGGCCTATGGCCTTTTTTTTTTGTGGGAAACCTTTCGCGGTATGGTATTAAAGC





GCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTC





GCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCAC





GTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCC





CAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCT





CCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATC





AACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAA





GCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTG





GATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTT





GATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGA





CTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCC





ATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAA





TCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAAC





AAACCATGCAAATGCTGAATGAGGGCATCGTTTCCACTGCGATGCTGGTTGCCAACGATC





AGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGAC





ATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACC





ACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTC





TCTCAGGGCCAGGCGGTTAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAA





AACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAAT





GCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGGTACCCGATAAAA





GCGGCTTCCTGACAGGAGGCCGTTTTGTTTC





Polynucleotide sequence of pYT-72Bg1 secreted yeast expression


vector:


(SEQ ID NO: 7)



TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACAAATATCATA






AAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGA





CTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCT





TTTTAACTGCATCTTCAATGGCTTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACAT





CATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAATCTGGAGC





GGAACCATGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCA





AGGACGCAGATGGCAACAAACCCAAGGAGCCTGGGATAACGGAGGCTTCATCGGAGAT





GATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAAC





TAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACTTTCAATGTAGGGAATTCGTT





CTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAACATTAGCTTTA





TCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCT





TGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCA





TCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTAACAACAACGAAGT





CAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCA





AAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTT





GTTCAGGTCTAACACTACCGGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACG





GCATCAGCCTTTTTGGAGGCTTCCAGCGCCTCATTTGGAAGTGGAACACCTGTAGCATCG





ATAGCAGCCCCCCCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCA





GAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCT





GGCAAAACGACGATTTTTTTAGGGGCAGACATTACAATGGTATATCCTTGAAATATATAT





AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCAGCTTCTCAATGATATTCGAATAC





GCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTACTT





GTTACCCATCATTGAATTTTGAACATCCGAACCTGGGAGTTTTCCCTGAAACAGATAGTA





TATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATTT





CGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCGG





TTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTGT





GCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCT





GAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAA





TCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAA





GAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAAC





AAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAA





CAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAA





CTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTT





CCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTG





CATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACT





TTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTT





TCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGA





TTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAA





CATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGT





AGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTT





TGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTT





TTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTT





CTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCC





GAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCG





TGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATG





CGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTA





TCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATG





CTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGA





TCATATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGA





TGAGTATACGTTGTCCTGGCCACGGCAGAAGCACGCTTATCGCTCCAATTTCCCACAACA





TTAGTCAACTCCGTTAGGCCCTTCATTGAAAGAAATGAGGTCATCAAATGTCTTCCAATG





TGAGATTTTGGGCCATTTTTTATAGCAAAGATTGAATAAGGCGCATTTTTCTTCAAAGCTT





TATTGTACGATCTGACTAAGTTATCTTTTAATAATTGGTATTCCTGTTTATTGCTTGAAGA





ATTGCCGGTCCTATTTACTCGTTTTAGGACTGGTTCAGAATTCCTCAAAAATTCATCCAAA





TATACAAGTGGATCGATGATAAGCTGTCAAACATGAGAATTCTTGAAGACGAAAGGGCC





TCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGG





TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCA





AATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGG





AAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCC





TTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG





GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC





GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTAT





TATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATG





ACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA





GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA





ACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAAC





TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA





CCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT





ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC





ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGA





GCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT





AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTG





AGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATAC





TTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGA





TAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT





AGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA





AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTC





TTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGT





AGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC





TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT





CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA





CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATG





AGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG





GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAG





TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG





CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG





CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCG





CCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTG





AGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATT





TCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAG





TATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACAC





CCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGA





CCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGC





AGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCAT





CCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGG





CCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCT





GTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTG





ATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATG





CGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGT





AGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGC





AGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCAT





GTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATC





GGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGAC





AGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGT





GCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATT





CACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAG





GTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGC





GGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGC





TCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTG





GTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGAC





AGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGG





GAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCG





CCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACG





AAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGT





CGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTC





CTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGC





GCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGGATCTGG





GCAAAACGTAGGGGCAAACAAACGGAAAAATCGTTTCTCAAATTTTCTGATGCCAAGAA





CTCTAACCAGTCTTATCTAAAAATTGCCTTATGATCCGTCTCTCCGGTTACAGCCTGTGTA





ACTGATTAATCCTGCCTTTCTAATCACCATTCTAATGTTTTAATTAAGGGATTTTGTCTTC





ATTAACGGCTTTCGCTCATAAAAATGTTATGACGTTTTGCCCGCAGGCGGGAAACCATCC





ACTTCACGAGACTGATCTCCTCTGCCGGAACACCGGGCATCTCCAACTTATAAGTTGGAG





AAATAAGAGAATTTCAGATTGAGAGAATGAAAAAAAAAAAAAAAAAAAGGCAGAGGAG





AGCATAGAAATGGGGTTCACTTTTTGGTAAAGCTATAGCATGCCTATCACATATAAATAG





AGTGCCAGTAGCGACTTTTTTCACACTCGAAATACTCTTACTACTGCTCTCTTGTTGTTTT





TATCACTTCTTGTTTCTTCTTGGTAAATAGAATATCAAGCTACAAAAAGCATACAATCAA





CTATCAACTATTAACTATATCGTAATACACAGGATCCACCATGAAGGCTGCTGCGCTTTC





CTGCCTCTTCGGCAGTACCCTTGCCGTTGCAGGCGCCATTGAATCGAGAAAGGTTCACCA





GAAGCCCCTCGCGAGATCTGAACCTTTTTACCCGTCGCCATGGATGAATCCCAACGCCAT





CGGCTGGGCGGAGGCCTATGCCCAGGCCAAGTCCTTTGTCTCCCAAATGACTCTGCTAGA





GAAGGTCAACTTGACCACGGGAGTCGGCTGGGGGGAGGAGCAGTGCGTCGGCAACGTG





GGCGCGATCCCTCGCCTTGGACTTCGCAGTCTGTGCATGCATGACTCCCCTCTCGGCGTG





CGAGGAACCGACTACAACTCAGCGTTCCCCTCTGGCCAGACCGTTGCTGCTACCTGGGAT





CGCGGTCTGATGTACCGTCGCGGCTACGCAATGGGCCAGGAGGCCAAAGGCAAGGGCAT





CAATGTCCTTCTCGGACCAGTCGCCGGCCCCCTTGGCCGCATGCCCGAGGGCGGTCGTAA





CTGGGAAGGCTTCGCTCCGGATCCCGTCCTTACCGGCATCGGCATGTCCGAGACGATCAA





GGGCATTCAGGATGCTGGCGTCATCGCTTGTGCGAAGCACTTTATTGGAAACGAGCAGG





AGCACTTCAGACAGGTGCCAGAAGCCCAGGGATACGGTTACAACATCAGCGAAACCCTC





TCCTCCAACATTGACGACAAGACCATGCACGAGCTCTACCTTTGGCCGTTTGCCGATGCC





GTCCGGGCCGGCGTCGGCTCTGTCATGTGCTCGTACAACCAGGGCAACAACTCGTACGCC





TGCCAGAACTCGAAGCTGCTGAACGACCTCCTCAAGAACGAGCTTGGGTTTCAGGGCTTC





GTCATGAGCGACTGGTGGGCACAGCACACTGGCGCAGCAAGCGCCGTGGCTGGTCTCGA





TATGTCCATGCCGGGCGACACCATGGTCAACACTGGCGTCAGTTTCTGGGGCGCCAATCT





CACCCTCGCCGTCCTCAACGGCACAGTCCCTGCCTACCGTCTCGACGACATGTGCATGCG





CATCATGGCCGCCCTCTTCAAGGTCACCAAGACCACCGACCTGGAACCGATCAACTTCTC





CTTCTGGACCCGCGACACTTATGGCCCGATCCACTGGGCCGCCAAGCAGGGCTACCAGG





AGATTAATTCCCACGTTGACGTCCGCGCCGACCACGGCAACCTCATCCGGAACATTGCCG





CCAAGGGTACGGTGCTGCTGAAGAATACCGGCTCTCTACCCCTGAACAAGCCAAAGTTC





GTGGCCGTCATCGGCGAGGATGCTGGGCCGAGCCCCAACGGGCCCAACGGCTGCAGCGA





CCGCGGCTGTAACGAAGGCACGCTCGCCATGGGCTGGGGATCCGGCACAGCCAACTATC





CGTACCTCGTTTCCCCCGACGCCGCGCTCCAGGCGCGGGCCATCCAGGACGGCACGAGG





TACGAGAGCGTCCTGTCCAACTACGCCGAGGAAAATACAAAGGCTCTGGTCTCGCAGGC





CAATGCAACCGCCATCGTCTTCGTCAATGCCGACTCAGGCGAGGGCTACATCAACGTGG





ACGGTAACGAGGGCGACCGTAAGAACCTGACTCTCTGGAACAACGGTGATACTCTGGTC





AAGAACGTCTCGAGCTGGTGCAGCAACACCATCGTCGTCATCCACTCGGTCGGCCCGGTC





CTCCTGACCGATTGGTACGACAACCCCAACATCACGGCCATTCTCTGGGCTGGTCTTCCG





GGCCAGGAGTCGGGCAACTCCATCACCGACGTGCTTTACGGCAAGGTCAACCCCGCCGC





CCGCTCGCCCTTCACTTGGGGCAAGACCCGCGAAAGCTATGGCGCGGACGTCCTGTACA





AGCCGAATAATGGCAATTGGGCGCCCCAACAGGACTTCACCGAGGGCGTCTTCATCGAC





TACCGCTACTTCGACAAGGTTGACGATGACTCGGTCATCTACGAGTTCGGCCACGGCCTG





AGCTACACCACCTTCGAGTACAGCAACATCCGCGTCGTCAAGTCCAACGTCAGCGAGTA





CCGGCCCACGACGGGCACCACGATTCAGGCCCCGACGTTTGGCAACTTCTCCACCGACCT





CGAGGACTATCTCTTCCCCAAGGACGAGTTCCCCTACATCCCGCAGTACATCTACCCGTA





CCTCAACACGACCGACCCCCGGAGGGCCTCGGGCGATCCCCACTACGGCCAGACCGCCG





AGGAGTTCCTCCCGCCCCACGCCACCGATGACGACCCCCAGCCGCTCCTCCGGTCCTCGG





GCGGAAACTCCCCCGGCGGCAACCGCCAGCTGTACGACATTGTCTACACAATCACGGCC





GACATCACGAATACGGGCTCCGTTGTAGGCGAGGAGGTACCGCAGCTCTACGTCTCGCT





GGGCGGTCCCGAGGATCCCAAGGTGCAGCTGCGCGACTTTGACAGGATGCGGATCGAAC





CCGGCGAGACGAGGCAGTTCACCGGCCGCCTGACGCGCAGAGATCTGAGCAACTGGGAC





GTCACGGTGCAGGACTGGGTCATCAGCAGGTATCCCAAGACGGCATATGTTGGGAGGAG





CAGCCGGAAGTTGGATCTCAAGATTGAGCTTCCTTGATAAGTCGACCTCGACTTTGTTCC





CACTGTACTTTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGT





TTTCCCATGTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATAT





AGCTATTCACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATT





TCAATTTGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGA





ATCGAATCCTAAGAGAATTGGATCTGATCCACAGGACGGGTGTGGTCGCCATGATCGCG





TAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGGCCAAAGCGGTC





GGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATATAGCGCTAGCA





GCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAGAGGCCCGGC





AGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGAC





GATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGA





TAAACTACCGCATTAAAGCTTTTTCTTTCCAATTTTTTTTTTTTCGTCATTATAAAAATCAT





TACGACCGAGATTCCCGGGTAATAACTGATATAATTAAATTGAAGCTCTAATTTGTGAGT





TTAGTATACATGCATTTACTTATAATACAGTTTTTTAGTTTTGCTGGCCGCATCTTCTCAA





ATATGCTTCCCAGCCTGCTTTTCTGTAACGTTCACCCTCTACCTTAGCATCCCTTCCCTTTG





CAAATAGTCCTCTTCCAACAATAATAATGTCAGATCCTGTAGAGACCACATCATCCACGG





TTCTATACTGTTGACCCAATGCGTCTCCCTTGTCATCTAAACCCACACCGGGTGTCATAAT





CAACCAATCGTAACCTTCATCTCTTCCACCCATGTCTCTTTGAGCAATAAAGCCGATAAC





AAAATCTTTGTCGCTCTTCGCAATGTCAACAGTACCCTTAGTATATTCTCCAGTAGATAG





GGAGCCCTTGCATGACAATTCTGCTAACATCAAAAGGCCTCTAGGTTCCTTTGTTACTTCT





TCTGCCGCCTGCTTCAAACCGCTAACAATACCTGGGCCCACCACACCGTGTGCATTCGTA





ATGTCTGCCCATTCTGCTATTCTGTATACACCCGCAGAGTACTGCAATTTGACTGTATTAC





CAATGTCAGCAAATTTTCTGTCTTCGAAGAGTAAAAAATTGTACTTGGCGGATAATGCCT





TTAGCGGCTTAACTGTGCCCTCCATGGAAAAATCAGTCAAGATATCCACATGTGTTTTTA





GTAAACAAATTTTGGGACCTAATGCTTCAACTAACTCCAGTAATTCCTTGGTGGTACGAA





CATCCAATGAAGCACACAAGTTTGTTTGCTTTTCGTGCATGATATTAAATAGCTTGGCAG





CAACAGGACTAGGATGAGTAGCAGCACGTTCCTTATATGTAGCTTTCGACATGATTTATC





TTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGT





TTCTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCT





TCCTTCTGTTCGGAGATTACCGAATCAAAAAAATTTCAAGGAAACCGAAATCAAAAAAA





AGAATAAAAAAAAAATGATGAATTGAAAAGCTTATCGATCCTACCCCTTGCGCTAAAGA





AGTATATGTGCCTACTAACGCTTGTCTTTGTCTCTGTCACTAAACACTGGATTATTACTCC





CAGATACTTATTTTGGACTAATTTAAATGATTTCGGATCAACGTTCTTAATATCGCTGAAT





CTTCCACAATTGATGAAAGTAGCTAGGAAGAGGAATTGGTATAAAGTTTTTGTTTTTGTA





AATCTCGAAGTATACTCAAACGAATTTAGTATTTTCTCAGTGATCTCCCAGATGCTTTCAC





CCTCACTTAGAAGTGCTTTAAGCATTTTTTTACTGTGGCTATTTCCCTTATCTGCTTCTTCC





GATGATTCGAACTGTAATTGCAAACTACTTACAATATCAGTGATATCAGATTGATGTTTT





TGTCCATAGTAAGGAATAATTGTAAATTCCCAAGCAGGAATCAATTTCTTTAATGAGGCT





TCCAGAATTGTTGCTTTTTGCGTCTTGTATTTAAACTGGAGTGATTTATTGACAATATCGA





AACTCAGCGAATTGCTTATGATAGTATTATAGCTCATGAATGTGGCTCTCTTGATTGCTGT





TCCGTTATGTGTAATCATCCAACATAAATAGGTTAGTTCAGCAGCACATAATGCTATTTT





CTCACCTGAAGGTCTTTCAAACCTTTCCACAAACTGACGAACAAGCACCTTAGGTGGTGT





TTTACATAATATATCAAATTGTGGCATGCTTAGCGCCGATCTTGTGTGCAATTGATATCTA





GTTTCAACTACTCTATTTATCTTGTATCTTGCAGTATTCAAACACGCTAACTCGAAAAACT





AACTTTAATTGTCCTGTTTGTCTCGCGTTCTTTCGAAAAATGCACCGGCCGCGCATTATTT





GTACTGCGAAAATAATTGGTACTGCGGTATCTTCATTTCATATTTTAAAAATGCACCTTTG





CTGCTTTTCCTTAATTTTTAGACGGCCCGCAGGTTCGTTTTGCGGTACTATCTTGTGATAA





AAAGTTGTTTTGACATGTGATCTGCACAGATTTTATAATGTAATAAGCAAGAATACATTA





TCAAACGAACAATACTGGTAAAAGAAAACCAAAATGGACGACATTGAAACAGCCAAGA





ATCTGACGGTAAAAGCACGTACAGCTTATAGCGTCTGGGATGTATGTCGGCTGTTTATTG





AAATGATTGCTCCTGATGTAGATATTGATATAGAGAGTAAACGTAAGTCTGATGAGCTAC





TCTTTCCAGGATATGTCATAAGGCCCATGGAATCTCTCACAACCGGTAGGCCGTATGGTC





TTGATTCTAGCGCAGAAGATTCCAGCGTATCTTCTGACTCCAGTGCTGAGGTAATTTTGC





CTGCTGCGAAGATGGTTAAGGAAAGGTTTGATTCGATTGGAAATGGTATGCTCTCTTCAC





AAGAAGCAAGTCAGGCTGCCATAGATTTGATGCTACAGAATAACAAGCTGTTAGACAAT





AGAAAGCAACTATACAAATCTATTGCTATAATAATAGGAAGATTGCCCGAGAAAGACAA





GAAGAGAGCTACCGAAATGCTCATGAGAAAAATGGATTGTACACAGTTATTAGTCCCAC





CAGCTCCAACGGAAGAAGATGTTATGAAGCTCGTAAGCGTCGTTACCCAATTGCTTACTT





TAGTTCCACCAGATCGTCAAGCTGCTTTAATAGGTGATTTATTCATCCCGGAATCTCTAA





AGGATATATTCAATAGTTTCAATGAACTGGCGGCAGAGAATCGTTTACAGCAAAAAAAG





AGTGAGTTGGAAGGAAGGACTGAAGTGAACCATGCTAATACAAATGAAGAAGTTCCCTC





CAGGCGAACAAGAAGTAGAGACACAAATGCAAGAGGAGCATATAAATTACAAAACACC





ATCACTGAGGGCCCTAAAGCGGTTCCCACGAAAAAAAGGAGAGTAGCAACGAGGGTAA





GGGGCAGAAAATCACGTAATACTTCTAGGGTATGATCCAATATCAAAGGAAATGATAGC





ATTGAAGGATGAGACTAATCCAATTGAGGAGTGGCAGCATATAGAACAGCTAAAGGGTA





GTGCTGAAGGAAGCATACGATACCCCGCATGGAATGGGATAATATCACAGGAGGTACTA





GACTACCTTTCATCCTACATAAATAGACGCATATAAGTACGCATTTAAGCATAAACACGC





ACTATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACG





TGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGCGCTCGTTTTCGGA





AACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAG





CGCTTTTGAAAACCAAAAGCGCTCTGAAGACGCACTTTCAAAAAACCAAAAACGCACCG





GACTGTAACGAGCTACTAAAATATTGCGAATACCGCTTCCACAAACATTGCTCAAAAGTA





TCTCTTTGCTATATATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCGCTCC





TTGAACTTGCATCTAAACTCGACCTCTACATCAACAGGCTTCCAATGCTCTTCAAATTTTA





CTGTCAAGTAGACCCATACGGCTGTAATATGCTGCTCTTCATAATGTAAGCTTATCTTTAT





CGAATCGTGTGAAAAACTACTACCGCGATAAACCTTTACGGTTCCCTGAGATTGAATTAG





TTCCTTTAGTATATGATACAAGACACTTTTGAACTTTGTACGACGAATTTTGAGGTTCGCC





ATCCTCTGGCTATTTCCAATTATCCTGTCGGCTATTATCTCCGCCTCAGTTTGATCTTCCGC





TTCAGACTGCCATTTTTCACATAATGAATCTATTTCACCCCACAATCCTTCATCCGCCTCC





GCATCTTGTTCCGTTAAACTATTGACTTCATGTTGTACATTGTTTAGTTCACGAGAAGGGT





CCTCTTCAGGCGGTAGCTCCTGATCTCCTATATGACCTTTATCCTGTTCTCTTTCCACAAA





CTTAGAAATGTATTCATGAATTATGGAGCACCTAATAACATTCTTCAAGGCGGAGAAGTT





TGGGCCAGATGCCCAATATGCTTGACATGAAAACGTGAGAATGAATTTAGTATTATTGTG





ATATTCTGAGGCAATTTTATTATAATCTCGAAGATAAGAGAAGAATGCAGTGACCTTTGT





ATTGACAAATGGAGATTCCATGTATCTAAAAAATACGCCTTTAGGCCTTCTGATACCCTT





TCCCCTGCGGTTTAGCGTGCCTTTTACATTAATATCTAAACCCTCTCCGATGGTGGCCTTT





AACTGACTAATAAATGCAACCGATATAAACTGTGATAATTCTGGGTGATTTATGATTCGA





TCGACAATTGTATTGTACACTAGTGCAGGATCAGGCCAATCCAGTTCTTTTTCAATTACC





GGTGTGTCGTCTGTATTCAGTACATGTCCAACAAATGCAAATGCTAACGTTTTGTATTTCT





TATAATTGTCAGGAACTGGAAAAGTCCCCCTTGTCGTCTCGATTACACACCTACTTTCATC





GTACACCATAGGTTGGAAGTGCTGCATAATACATTGCTTAATACAAGCAAGCAGTCTCTC





GCCATTCATATTTCAGTTATTTTCCATTACAGCTGATGTCATTGTATATCAGCGCTGTAAA





AATCTATCTGTTACAGAAGGTTTTCGCGGTTTTTATAAACAAAACTTTCGTTACGAAATC





GAGCAATCACCCCAGCTGCGTATTTGGAAATTCGGGAAAAAGTAGAGCAACGCGAGTTG





CATTTTTTACACCATAATGCATGATTAACTTCGAGAAGGGATTAAGGCTAATTTCACTAG





TATGTTTCAAAAACCTCAATCTGTCCATTGAATGCCTTATAAAACAGCTATAGATTGCAT





AGAAGAGTTAGCTACTCAATGCTTTTTGTCAAAGCTTACTGATGATGATGTGTCTACTTTC





AGGCGGGTCTGTAGTAAGGAGAATGACATTATAAAGCTGGCACTTAGAATTCCACGGAC





TATAGACTATACTAGTATACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCC





TTTAACGAGGCCTTACCACTCTTTTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTG





ATCTAAGATTCTATCTTCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAA





ATGCAAAAGGCACTTCTACAATGGCTGCCATCATTATTATCCGATGTGACGCTGCA





Polynucleotide sequence of Variant No. 73 yCDS:


(SEQ ID NO: 8)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG





GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA





CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA





TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC





TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC





CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT





AAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACT





GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polynucleotide sequence of Variant No. 73:


(SEQ ID NO: 9)



CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT






GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG





AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC





ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC





TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA





GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG





ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA





TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC





CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC





CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC





TGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA





GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA





TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA





TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT





CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC





AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG





GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT





GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT





GTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCC





CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT





T





Polypeptide sequence of Variant No. 73:


(SEQ ID NO: 10)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG





LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG





DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF





YEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of Variant No. 218 yCDS:


(SEQ ID NO: 11)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG





GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA





CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA





TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC





TGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC





CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT





AAGAGAAAGTTGGGTTTCTATAACTGGACATCTAGGCTAAAAAGTCACATTAATCCTACT





GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polynucleotide sequence of Variant No. 218 hCDS:


(SEQ ID NO: 12)



CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT






GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG





AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC





ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC





TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA





GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG





ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA





TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC





CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC





CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC





TGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA





GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA





TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA





TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT





CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC





AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG





GCTGTAGCTATTATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT





GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT





GTGAAAAGGAAGCTAGGGTTCTATAACTGGACTTCAAGGTTAAAAAGTCACATAAATCC





CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT





T





Polypeptide sequence of Variant No. 218:


(SEQ ID NO: 13)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG





LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG





DNFEVWERPLSGLAWAVAIINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFY





NVVTSRLKSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of Variant No. 326 yCDS:


(SEQ ID NO: 14)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAAGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT





GGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTT





ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA





ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG





CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG





CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT





TAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTAC





TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polypeptide sequence of Variant No. 326:


(SEQ ID NO: 15)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF





GLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRK





GDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF





YNVVTSRLKSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of Variant No. 206 yCDS:


(SEQ ID NO: 16)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG





GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA





CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA





TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC





TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC





CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT





AAGAGAAAGTTGGGTTTCTATAATTGGACCTCTAGGCTAAGAAGTCACATCAATCCTACT





GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polynucleotide sequence of Variant No. 206 hCDS:


(SEQ ID NO: 17)



CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT






GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG





AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC





ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC





TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA





GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG





ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA





TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC





CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC





CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC





TGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA





GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA





TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA





TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT





CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC





AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG





GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT





GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT





GTGAAAAGGAAGCTAGGGTTCTATAACTGGACTTCAAGGTTAAGAAGTCACATAAATCC





CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT





T





Polypeptide sequence of Variant No. 206:


(SEQ ID NO: 18)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG





LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG





DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF





YNWTSRLRSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of Variant No. 205 yCDS:


(SEQ ID NO: 19)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG





GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA





CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA





TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC





TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC





CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT





AAGAGAAAGTTGGGTTTCTATGATTGGGACTCTAGGCTAAGAAGTCACATCAATCCTACT





GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polynucleotide sequence of Variant No. 205 hCDS:


(SEQ ID NO: 20)



CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT






GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG





AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC





ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC





TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA





GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG





ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA





TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC





CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC





CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC





TGACATTGATGATTCCTGGGCGAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA





GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA





TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA





TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT





CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC





AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG





GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT





GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT





GTGAAAAGGAAGCTAGGGTTCTATGATTGGGATTCAAGGTTAAGAAGTCACATAAATCC





CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT





T





Polypeptide sequence of Variant No. 205:


(SEQ ID NO: 21)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG





LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG





DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF





YDWDSRLRSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of Variant No. 76 yCDS:


(SEQ ID NO: 22)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGAGGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATG





GCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTA





CTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAA





TTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGC





TGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGC





CTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTT





AAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACT





GGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polynucleotide sequence of Variant No. 76 hCDS:


(SEQ ID NO: 23)



CTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAGCGCTTCAT






GTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGG





AGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGC





ATTGATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCC





TCAGCGCTTTCCTCATGGGATTCGCCAGCTAGCTAATTATGTTCACAGCAAAGGACTGAA





GCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCTTCCCTGGGAGTTTTGG





ATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTTGA





TGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC





CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCC





CTTTCAAAAGCCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGC





TGACATTGATGATTCCTGGCGTAGTATAAAGAGTATCTTGGACTGGACATCTTTTAACCA





GGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATATGTTAGTGA





TTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCA





TGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCT





CCTTCAGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACC





AGCTTAGACAGGGAGACAACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGG





GCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCTTATACCATCGCAGTT





GCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCCT





GTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCC





CACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACT





T





Polypeptide sequence of Variant No. 76:


(SEQ ID NO: 24)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG





LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQG





DNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGF





YEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of Mfalpha signal peptide:


(SEQ ID NO: 25)



ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCT






Polypeptide sequence of Mfalpha signal peptide:


(SEQ ID NO: 26)



MRFPSIFTAVLFAASSALA






Polynucleotide sequence of MMO435:


(SEQ ID NO: 27)



ttaactatatcgtaatacacaggatccaccATGAGATTTCCTTCAATTTTTACTG






Polynucleotide sequence of MMO439:


(SEQ ID NO: 28)



AGTAGGTGTACGGGCTAACCCGTTATCCAAAGCTAATGCGGAGGATGC






Polynucleotide sequence of MMO514:


(SEQ ID NO: 29)



TTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTTTGGATAACGGGTTAGCCCG






Polynucleotide sequence of MMO481:


(SEQ ID NO: 30)



GAGCTAAAAGTACAGTGGGAACAAAGTCGAGGTCGACTTATAACAAATCTTTCAAAGAC






A





Polynucleotide sequence of Synthetic mammalian signal peptide:


(SEQ ID NO: 31)



ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCC






Polynucleotide sequence of LAKE Fw:


(SEQ ID NO. 32)



CGATCGAAGCTTCGCCACCA






Polynucleotide sequence of Br reverse:


(SEQ ID NO: 33)



CTTGCCAATCCATTGTCCAGGGAGTGGACACCAGTCGTTA






Polynucleotide sequence of Br Fw:


(SEQ ID NO: 34)



TAACGACTGGTGTCCACTCCCTGGACAATGGATTGGCAAG






Polynucleotide sequence of hGLA Rv:


(SEQ ID NO: 35)



CGATCGGCGGCCGCTCAAAGTAAGTCTTTTAATGACA






Polynucleotide sequence of SP-GLA (yCDS):


(SEQ ID NO: 36)



ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTTTGG






ATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTA





ACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATG





GCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGA





TGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGA





GATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTA





GGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTAC





TATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGA





TGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTA





AACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTT





CAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGA





CATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGA





AAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGG





GAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGC





CGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACT





TCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATT





GAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTG





TTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCT





CTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAA





GAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACTGG





TACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polynucleotide Sequence of MFleader-GLA (yCDS):


(SEQ ID NO: 37)



ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC






CAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT





TACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT





AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA





TCTTTGGATAAAAGATTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCAC





TGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGA





GAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTT





ATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGT





TACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACA





GCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCC





CAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATT





TGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAAC





ACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCT





TGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATT





GGCGTAACTTTGCTGACATAGATGATTCATGGAAGTCAATCAAATCTATCTTGGATTGGA





CTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTG





ATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTT





TGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCC





AAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTA





AACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCT





GGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTA





CACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTAC





ACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAG





TCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTT





GAAAGATTTGTTA





Polypeptide Sequence of MFleader:


(SEQ ID NO: 38)



MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLL






FINTTIASIAAKEEGVSLDKR





Polynucleotide sequence of Variant No. 395 yCDS:


(SEQ ID NO: 39)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT





GGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTT





ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA





ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG





CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG





CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT





TAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTAC





TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polypeptide sequence of Variant No. 395:


(SEQ ID NO: 40)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF





GLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRK





GDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF





YNVVTSRLKSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of Variant No. 402 yCDS:


(SEQ ID NO: 41)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACGGATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAAGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT





GGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTT





ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA





ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG





CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG





CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT





TAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTAC





TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA





Polypeptide sequence of Variant No. 402:


(SEQ ID NO: 42)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF





GLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRK





GDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF





YNVVTSRLKSHINPTGTVLLQLENTMQMSLKDLL





Polynucleotide sequence of Variant No. 625 yCDS:


(SEQ ID NO: 43)



TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGA





GATGGCTGAACGGATGGTAACCGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT





GGCCGCACCCCTATTCATGTCTAATGATCTACGTGCGATATCACCCCAAGCAAAGGCTTT





ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA





ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG





CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG





CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT





TAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTAC





TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAACCTCTTTGAAAGATTTGTTA





Polypeptide sequence of Variant No. 625:


(SEQ ID NO: 44)



LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVTEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF





GLSWDQQVTQMALWAIMAAPLFMSNDLRAISPQAKALLQDKDVIAINQDPLGKQGYQLRK





GDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF





YNVVTSRLKSHINPTGTVLLQLENTMQTSLKDLL





Polynucleotide sequence of Variant No. 648 yCDS:


(SEQ ID NO: 45)



TTGGATAACGGGTTAGCCCGTACACCTCCGATGGGTTGGCTTCACTGGGAAAGATTCATG






TGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCGAAGA





GATGGCTGAACGGATGGTAACCGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTA





TTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCC





AGAGATTCCCACATGGCATACGTCAGCTTGCAAACCATGTACACAGCAAAGGTCTAAAG





TTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGT





TACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGAT





GGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCT





CTAAACAGGACTGGTAGGCCGATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCG





TTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCT





GACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTCGTAACCAG





GAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATA





GGGAACTTTGGGCTATCATGGGACCAACAAGTTACACAAATGGCTTTGTGGGCGATCAT





GGCCGGCCCCCTATTCATGTCTAATGATCTACGTGCGATATCACCCCAAGCAAAGGCTTT





ACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCA





ATTGAGAAAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGAGATGCGTGGG





CTGTTGCTATTATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCCCGGTAG





CCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGT





TAAGAGACAATTGGGTTTCTATAACGCAACCTCTAGGCTAAAAAGTCACATTAATCCTAC





TGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAACCTCTTTGAAAGATTTGTTA





Polypeptide sequence of Variant No. 648:


(SEQ ID NO: 46)



LDNGLARTPPMGWLHWERFMCNLDCQEEPDSCISEKLFEEMAERMVTEGWKDAGYEYLCI






DDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYY





DIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQ





KPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNF





GLSWDQQVTQMALWAIMAGPLFMSNDLRAISPQAKALLQDKDVIAINQDPLGKQGYQLRK





GDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGF





YNATSRLKSHINPTGTVLLQLENTMQTSLKDLL






Example 1
GLA Gene Acquisition and Construction of Expression Vectors

A synthetic gene coding for a WT human GLA was designed for optimized gene expression in Saccharomyces cerevisiae (SEQ ID NO:3), assembled, and subcloned into the E. coli expression vector pCK100900i (SEQ ID NO:6).


A chimeric GLA expression construct encoding a 19 amino acid S. cerevisae MFalpha signal peptide fused to the mature form of yeast-optimized GLA was generated in a yeast expression vector designed for secreted expression, as follows. A fragment coding for the MFalpha signal peptide (SEQ ID NO:25) was amplified by PCR using the oligonucleotides MM0435 (SEQ ID NO:27) and MM0439 (SEQ ID NO:28) from S288C genomic DNA, and a fragment coding for a synthetic GLA (SEQ ID NO:3) was amplified using primers MM0514 (SEQ ID NO:29) and MM0481 (SEQ ID NO:30). Additional sequence at the 5′ ends of these oligonucleotides provide homology for yeast recombination cloning when cotransformed with linearized plasmid pYT-72Bgl (SEQ ID NO:7). In the resulting vector, the expression of fusion protein SP-GLA (SEQ ID NO:36) is driven by the ADH2 promoter. A fusion construct encoding a fusion of an 83 amino acid MFalpha leader peptide (SEQ ID NO:38) N-terminally fused to GLA (SEQ ID NO:37) was cloned using the same techniques. Recombination cloning and gene expression were performed in S. cerevisiae strain INVSc1. Directed evolution techniques generally known by those skilled in the art were used to generate libraries of gene variants from this plasmid construct (See e.g., U.S. Pat. No. 8,383,346 and WO2010/144103).


A chimeric GLA expression construct encoding a synthetic signal peptide fused to a synthetic gene coding for the mature human GLA coding sequence for secreted expression in transient transfections was generated as follows. Oligonucleotides PLEV113Fw (SEQ ID NO:32) and SPGLARv (SEQ ID NO:33) were used to amplify a fragment coding for a synthetic signal peptide (SEQ ID NO:31) using PCR. A second fragment coding for the native human coding sequence for the mature form of GLA (SEQ ID NO:4) was amplified using oligonucleotides SPGLAFw (SEQ ID NO:34) and GLARv (SEQ ID NO:35). Splicing by Overlap Extension PCR was used to recombine these fragments, and the resulting chimeric fragment was ligated into the HindIII/Not I linearized mammalian expression vector pLEV113. Directed evolution techniques generally known by those skilled in the art were used to generate specific gene variants from this plasmid construct.


Example 2
High-Throughput Growth and Assays
High-Throughput (HTP) Growth of GLA and GLA Variants

Yeast (INVSc1) cells transformed with vectors expressing GLA and GLA variants using the lithium acetate method were selected on SD-Ura agar plates. After 72 h incubation at 30° C. colonies were placed into the wells of Axygen® 1.1 ml 96-well deep well plates filled with 200 μl/well SD-Ura broth (2 g/L SD-Ura, 6.8 g/L yeast nitrogen base without amino acids [Sigma Aldrich]), 3.06 g/L sodium dihydrogen phosphate, 0.804 g/L disodium hydrogen phosphate, pH 6.0 supplemented with 6% glucose. The cells were allowed to grow for 20-24 hours in a Kuhner shaker (250 rpm, 30° C., and 85% relative humidity). Overnight culture samples (20 μL) were transferred into Corning Costar® 96-well deep plates filled with 380 μL of SD-ura broth supplemented with 2% glucose. The plates were incubated for 66-84 h in a Kuhner shaker (250 rpm, 30° C., and 85% relative humidity). The cells were then pelleted (4000 rpm×20 min), and the supernatants isolated and stored at 4° C. prior to analysis.


HTP-Analysis of Supernatants

GLA variant activity was determined by measuring the hydrolysis of 4-methylumbelliferyl α-D-galactopyranoside (MUGal). For this assay, 5-50 μL of yeast culture supernatant produced as described above, was mixed with 0-45 μL of McIlvaine Buffer (McIlvaine, J. Biol. Chem., 49:183-186 [1921]), pH 4.8 and 50 μL of 2 mM MUGal in 50 mM citrate, 200 mM KCl, pH 4.6 in a 96-well, black, opaque bottom plate. The reactions were mixed briefly and incubated at 37° C. for 30-180 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm).


HTP-Analysis of Supernatants Pretreated with Acid


GLA variants were challenged with acidic buffer to simulate the extreme pHs that the variants may encounter in lysosomes. First, 50 μL of yeast culture supernatant and 50 uL of McIlvaine buffer (pH 3.3-4.3) were added to the wells of a 96-well round bottom plate. The plates were sealed with a PlateLoc Thermal Microplate Sealer (Agilent) and incubated at 37° C. for 1-3 h. For the assay, 10-50 μL of acid-pH-challenged sample was mixed with 0-40 μL of McIlvaine buffer pH 4.8, 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37° C. for 30-180 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm).


HTP-Analysis of Supernatants Pretreated with Base


GLA variants were challenged with basic (neutral) buffer to simulate the pHs that the variants encounter in the blood following their administration to a patient. First, 50 μL of yeast culture supernatant and 50 uL of McIlvaine buffer (pH 7.0-8.2) or 200 mM sodium bicarbonate (pH 9.1-9.7) were added to the wells of a 96-well round bottom plate. The plates were sealed and incubated at 37° C. for 1-18 h. For the assay, 10-50 μL of basic-pH-challenged sample was mixed with 0-40 μL of McIlvaine buffer pH 4.8, 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37° C. for 30-180 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm).


HTP-Analysis of Supernatants Pretreated with Bovine Serum


GLA variants were challenged with bovine serum to simulate the conditions the variants encounter following infusion into a patient. First, 20 μL of yeast culture supernatant and 80 μL of bovine serum were added to the wells of a 96-well round bottom plate. The plates were sealed and incubated at 37° C. for 1 h. For the assay, 50 μL of serum-challenged sample was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37° C. for 180 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm).









TABLE 2.1







Relative Activity of GLA Variants After No Challenge


(NC) or Challenge at the Indicated pH1, 2












Variant

pH
pH

SEQ ID


#
NC
4.3
7.0
Amino Acid Differences Relative to SEQ ID NO: 5
NO:















1
+
+
+
A337S
47


2
+
+
+
E43D
48


3
+
+
+
E43D/E48D
49


4
+
+++
+
E43D/E48D/I208V/N247D/Q299R/Q302K/R373K/I376V
50


5
++
++
++
E43D/E48D/I208V/R373K
51


6
+
+++
+
E43D/E48D/I208V/R373K/I376V
52


7
+
++
+
E43D/E48D/N247D/Q299R/Q302K/R373K/I376V
53


8
++
+++
+++
E43D/E48D/N247D/Q302K/R373K
54


9
+
+++
+
E43D/E48D/Q302K/R373K/I376V
55


10
++
+++
++
E43D/I208V/N247D
56


11
+
+++
++
E43D/I208V/N247D/Q299R/R373K/I376V
57


12
+
++
+
E43D/I208V/Q299R/R373K/I376V
58


13
++
+++
++
E43D/N247D/R373K/I376V
59


14
+
+++
++
E43D/R373K/I376V
60


15
+
+
+
E48D/I208V/Q299R/Q302K/R373K
61


16
+
++
+
E48D/R373K/I376V
62


17
+
+
+
E48G/R373K
63


18
+
+
++
F217S
64


19
+
++
+
I208V/N247D/Q299R/Q302K/R373K/I376V
65


20
+
+++
++
I208V/N247D/Q299R/R373K/I376V
66


21
+
+++
++
I208V/N247D/R373K/I376V
67


22
+
+
+
I208V/Q299R/I376V
68


23
+
+++
++
I208V/Q302K/R373K/I376V
69


24
+
++
+
I376V
70


25
+
+
+
K36Q
71


26
+
+
+
P179S/R373K
72


27
+
+
+
Q299R/M322V/R373K
73


28
+
+
+
Q299R/Q302K/R373K
74


29
+
+
+
Q299R/Q302K/R373K/I376V
75


30
+
++
+
Q302K/I376V
76


31
+
+
+
R373K
77


32
+
++
+
R373K/I376V
78






1Relative activity was calculated as activity of the variant/activity of WT GLA (SEQ ID NO: 5 (encoded by SEQ ID NO: 3).




2+ = 0.5 to 1.5 relative activity over WT GLA (SEQ ID NO: 5); ++ = >1.5 to 2.5 relative activity over WT GLA (SEQ ID NO: 5); and +++ = >2.5 relative activity over WT GLA (SEQ ID NO: 5).














TABLE 2.2







Relative Activity of GLA Variants After No Challenge


(NC) or Challenge at the Indicated pH1, 2, 3












Variant

pH
pH

SEQ ID


#
NC
4.2
7.1
Amino Acid Differences Relative to SEQ ID NO: 5
NO:















33
+
+
+
A199H/E367S
79


34
+
++
++
A337P
80


35
++
++
++
A339S
81


36
+
++
++
A350G
82


37
+
+
+
D105A
83


38

+

D105S
84


39
+
++
++
D124N/E147G/N161K/R162Q/T163V/R165A/I167S/
85






V168I/Y169V/S170—/M177S/F217E


40
++
++
++
D396R
86


41
+
+
+
D396T
87


42
+++
+++
+++
E367N
88


43
+
+
+
E367T
89


44
+
++
+
E387K
90


45
++
++
++
E387Q
91


46
+
+++
+
E387R
92


47
+
++
+
E387T
93


48
+
+
+
E40D
94


49
+
+
+
F180R
95


50
++
++
++
F180S
96


51
++
++
+
F198S
97


52
++
+
++
F217D
98


53
+
++
++
F217R
99


54
+
+
+
F352I
100


55
++
+++
++
F352V/F365I
101


56
++
++
++
F365I
102


57
++
++
++
F365K
103


58
++
++
++
F365L
104


59
+
+
+
F365R
105


60
+
+
+
F365T
106


61
++
++
++
F365V
107


62
++
+
++
G303Q/R373V
108


63
+
+
++
H155A
109


64
+
+
++
H155L
110


65
+
+
+
H155R
111


66
+
+
+
H155T
112


67
++
+
++
H375E
113


68
+
++
+
H84S
114


69
+
+
+
I102L
115


70
+
+
+
I102L/L394V
116


71
++
+
++
I123T/T369N
117


72
+
+
+
I167V
118


73
+
+++
++
K206A
10


74
+
+++
++
K206M
119


75
+
+++
++
K206Q
120


76
+
+++
++
K206R
24


77
+
++
+
K206T/V359S
121


78
++
++
++
K343D
122


79
++
++
++
K343G
123


80
+
+
+
K362Q
124


81
+
+
+
K362R
125


82
+
+
+
K36D
126


83
+
++
+
K36E
127


84
++
++
++
K395*
128


85
+
+
+
K395G
129


86
++
++
++
K395P
130


87
+
++
+
K395R
131


88
+
++
+
K395S
132


89
++
+
+
K395T
133


90
+
+
+
K96I
134


91
+
+
++
K96L
135


92
+
+
+
K96R
136


93
++
+++
+
K96R/L397V
137


94
+
+
+
L100F
138


95
+
+
+
L158A
139


96
+
+
+
L158I
140


97
+
+
+
L158M
141


98
+
+
+
L158R
142


99
+
+
+
L23M
143


100
+
+
+
L23T
144


101
+++
+++
+++
L316D
145


102
+++
+++
+++
L316E
146


103
++
++
++
L384F
147


104
++
++
++
L386V
148


105
+++
++
++
L394A
149


106
++
++
+++
L394R
150


107
+++
+++
+++
L394S
151


108
+++
+++
+++
L394T
152


109
++
++
+++
L397*
153


110
+++
++
+++
L397D
154


111
++
++
++
L397H
155


112
+
++
+
L397I
156


113
++
+
+++
L397Q
157


114
++
++
++
L397R
158


115
++
++
+++
L397T
159


116
++
++
++
L398E
160


117
++
++
++
L398G
161


118
++
++
++
L398N
162


119
++
++
++
L398Q
163


120
++
++
++
L398R
164


121
+
++
++
L44R/L384F
165


122
++
++
++
L44T
166


123

+

M20D/Q302K
167


124
++
++
+
M253F
168


125
+
+
+
M322I
169


126
+++
+++
+++
M390D
170


127
++
++
++
M390R
171


128
+
+
+
M390T
172


129
+
++
++
M392G
173


130
+
++
+
M392P
174


131
++
+
++
M392S
175


132
+
+
+
M39Y
176


133
+
+
+
N388R
177


134
+
+
+
N91Q
178


135
++
+++
++
Q190S/T369D
179


136
+
+
+
Q249A
180


137
+
+
+
Q302A
181


138
++
++
++
Q385H
182


139
+
+
+
Q385I
183


140
++
++
++
Q385L
184


141
+
+
+
Q391G
185


142
+
+
+
Q80A
186


143
+
+
+
Q80H
187


144
+
++
+
Q80V
188


145
+
+
+
Q88A
189


146
+
+
+
Q88F
190


147
++
++
++
Q88H
191


148
+
++
+
Q88R
192


149
++
+
++
Q88S
193


150
+
+
+
R162H
194


151
+
+
+
R162S
195


152
++
++
++
R221K/A350G
196


153
+
+
++
R221T
197


154
++
+
++
R301I/K362T
198


155
+
+
+
R301L
199


156
++
+
++
R371S
200


157
++
+
++
R371V
201


158
++
+
++
R87K
202


159
+
+
+
R87P/L398R
203


160
+
++
+
S166A
204


161
+
+
+
S166H
205


162
+
+
+
S166K
206


163
+
+
+
S31D
207


164
+


S34D/M392P
208


165
+


S34G
209


166
++
+
+
S34H/M390R
210


167
+
+
+
S34R
211


168
++
++
++
S374M
212


169
++
++
++
S374T
213


170
++
++
++
S393E
214


171
++
++
++
S393G
215


172
+
+
+
S393H
216


173
++
++
++
S393P
217


174
+
+
+
S47I
218


175
+
++
+
S47R
219


176
+
+
+
S47T
220


177
+
++
+
S95D
221


178
++
+++
++
S95E
222


179
+
+
+
S95Q
223


180
++
++
+++
T369D
224


181
+
+
+
T369S
225


182
++
+
+
T389S
226


183
+
+
+
V133I
227


184
++
+
+
V168A
228


185
+
++
+
V168L
229


186
++
++
+++
V345N
230


187
+
+
+
V345Y
231


188
+
+
+
V359E
232


189
+
+
+
V93I
233


190
++
+
++
W178H
234


191
+
++
+
W178S
235






1Relative activity was calculated as activity of the variant/activity of WT GLA (SEQ ID NO: 5 (encoded by SEQ ID NO: 3).




2Variant # 73 (Rd2BB) has the polynucleotide sequence of SEQ ID NO: 8 and polypeptide sequence of SEQ ID NO: 10.




3− = <0.5 relative activity to WT GLA (SEQ ID NO: 5); + = 0.5 to 1.5 relative activity over WT GLA (SEQ ID NO: 5); ++ = >1.5 to 2.5 relative activity over WT GLA (SEQ ID NO: 5); and +++ = >2.5 relative activity over WT GLA (SEQ ID NO: 5).














TABLE 2.3







Relative Activity of GLA Variants After No Challenge


(NC) or Challenge at the Indicated pH












Variant

pH
pH
Amino Acid Differences
SEQ


#
NC
4.2
7.6
Relative to SEQ ID NO: 10
ID NO:















192
+
+
+
A206E
236


193
+
+
+
A206G
237


194
++
++
++
A206R
238


195
+
+
+
A206S
239


196
+
+
+
A350G
240


197
++
++
++
A350G/K362Q/T369A
241


198
++
++
+++
A350G/T369D
242


199
++
++
++
A350G/T369S
243


200
+
+
+
C143A
244


201
+
+
+
C143T
245


202
+
+
+
C59A
246


203
++
++
+++
E367A/T369D
247


204
+
+
+
E367D
248


205
++
++
++
E367D/T369D
21


206
+++
+++
+++
E367N
18


207
++
+++
+++
E367N/R373K
249


208
++
+++
+++
E367N/R373K/I376V
250


209
+
+
+
E367P/T369D
251


210
++
++
++
F365L/E367N
252


211
++
++
++
F365L/E367N/I376V
253


212
++
++
++
F365L/E367N/R373K/I376V
254


213
+


H15Q/
255


214
+++
+++
+++
K343D/F365L/E367N
256


215
+
+
+
K343G
257


216
++
+++
+++
K343G/F365L/E367N/R373K
258


217
++
++
++
L316D
259


218
+++
+++
+++
M322I/E367N/R373K
13


219
+
+
+
M322I/R373K
260


220
+
+
++
M322V/R373K/I376V
261


221
+
+
+
M390I
262


222
++
++
+
P228Q/T369D
263


223
+
++
++
Q302K/A337P/A350G/K362Q
264


224
++
+++
+++
Q302K/M322V/E367N
265


225
+
+
+
R165S
266


226
+
+
++
R221T/F365L
267


227



R325H
268


228
+
+
+
R373K
269


229
+

+
R373K/I376V
270


230
+

+
S374R
271


231
++
++
++
T369D
272


232
++
++
++
T369S
273





1. Relative activity was calculated as activity of the variant/activity of Rd2BB (SEQ ID NO: 10)


2. Variant # 218 (Rd3BB) has the polynucleotide sequence of SEQ ID NO: 11 and polypeptide sequence of SEQ ID NO: 13.


3. − = <0.5 relative activity to Rd2BB (SEQ ID NO: 10); + = 0.5 to 1.5 relative activity over Rd2BB (SEQ ID NO: 10); ++ = >1.5 to 2.5 relative activity over Rd2BB (SEQ ID NO: 10); and +++ = >2.5 relative activity over Rd2BB (SEQ ID NO: 10).













TABLE 2.4







Relative Activity of GLA Variants After No Challenge (NC)


or Challenge at the Indicated pH or Condition1, 2













Variant

pH
pH

Amino Acid Differences Relative to SEQ ID NO: 5
SEQ ID


#
NC
4.2
7.6
Serum
(WT GLA)
NO:
















233
+++
+++
+++
+++
K206A/F217R/N247D/L316D/A350G/E367D/T369D
274


234
+++
+++
+++
+++
K206A/F217R/N247D/Q302K/A350G/E367D/T369D
275


235
+++
+++
+++
+++
K206A/F217R/N247D/Q302K/L316D/A337P/A350G/
276







E367D/T369D


236
+++
++
+++
++
K206A/F217R/Q302K/E367D/T369D
277


237
+++
+++
+++
+++
K206A/F217R/Q302K/L316D/A337P/A350G/E367D/
278







T369D


238
++
+++
+++
++
K206A/I208V/M322V/K343G/F365L/R373K/I376V
279


239
+++
+++
+++
++
K206A/I208V/R221K/N247D/M322I/K343D/F365L/
280







R373K/I376V


240

+
+
+
K206A/L269I/P349L/R373K
281


241
++
+++
+++
++
K206A/N247D/M322V/K343D/R373K/I376V
282


242
+++
+++
+++
+++
K206A/N247D/M322V/K343G/F365L/R373K
283


243
+++
+++
+++
+++
K206A/N247D/Q302K/A337P/K343G/A350G
284


244
+++
+++
+++
+++
K206A/N247D/Q302K/L316D/A350G
285


245
++
+++
+++
++
K206A/N247D/Q302K/M322V/F365L/R373K/I376V
286


246
+++
+++
+++
+++
K206A/Q302K/L316D/A337P
287


247
+++
+++
+++
+++
K206A/R221K/N247D/M322V/K343D/R373K
288


248
+
+
+
+
K206A/R221T/M322V/K343G/R373K
289


249
++
++
+++
++
K206A/R221T/M322V/R373K
290


250
+
+
+
+
K96I/K206A/F217R
291


251
++
+
++
+
K96I/K206A/F217R/N247D
292


252
+++
+++
+++
+++
K96I/K206A/F217R/N247D/A350G/E367D/T369D
293


253
+++
+++
+++
+++
K961/K206A/F217R/N247D/Q302K/L316D/A337P/
294







E367D/T369D


254
+
+
+
+
L100F/K206A
295


255
+++
+++
+++
++
L100F/K206A/I208V/R221K/N247D/Q302K/M322I/
296







K343D/F365L/I376V


256
++
+++
+++
+++
L100F/K206A/I208V/R221K/N247D/Q302K/M322V/
297







K343D/F365L/I376V


257
++
++
++
++
L100F/K206A/I208V/R221T/N247D/K343D/F365L/
298







I376V


258
+++
++
+++
++
L100F/K206A/I208V/R221T/Q302K/M322I/K343D/
299







I376V


259
+
+
+
+
L100F/K206A/M322V/F365L/R373K/I376V
300


260
+
+
+
+
L100F/K206A/N247D/F365L/R373K/I376V
301


261
++
++
++
+
L100F/K206A/N247D/M322V/K343D/I376V
302


262
++
+++
+++
+++
L100F/K206A/R221K/N247D/Q302K/M322V/F365L/
303







R373K/I376V


263
+
++
++
+++
L100F/K206A/R221K/N247D/Q302K/M322V/I376V
304


264
++
++
+++
++
L100F/K206A/R221K/N247D/Q302K/M322V/K343D/
305







R373K/I376V


265
+
+
+
+
L100F/K206A/R221K/R373K/I376V
306


266
++
++
+++
++
L100F/K206A/R221T/M322I/K343E/F365L/R373K
307


267
+++
+++
+++
+++
L100F/K206A/R221T/N247D/Q302K/K343D/F365L/
308







R373K


268
+
+
+
+
L100F/K206A/R373K/I376V
309


269

+
+
+
L37I/K206A/R221K/N247D/M322I/R373K
310


270
+
+
+
+
L44R/C143Y/K206A/A337P/A350G
311


271

+
+
+
L44R/E187G/K206A/A337P/A350G
312


272
+
+
+
+
L44R/K206A
313


273
+
+
+
+
L44R/K206A/E367D/T369D
314


274
+
+
+
+
L44R/K206A/F217R/A350G
315


275
++
++
++
++
L44R/K206A/F217R/N247D/A337P
316


276
+++
+++
+++
+++
L44R/K206A/F217R/N247D/L316D/A337P/A350G/
317







E367D/T369D


277
+++
+++
+++
+++
L44R/K206A/F217R/N247D/L316D/A337P/E367D/
318







T369D


278
+++
+++
+++
+++
L44R/K206A/F217R/N247D/L316D/A350G/E367D/
319







T369D


279
++
+++
+++
+++
L44R/K206A/F217R/N247D/Q302K/A350G
320


280
+
+
+
+
L44R/K206A/F217R/Q302K/E367D/T369D
321


281
+
+
+
+
L44R/K206A/I208V/R221K/M322V/K343D/F365L/
322







R373K


282
+
+
+
+
L44R/K206A/N247D/A337P
323


283
+++
+++
+++
+++
L44R/K206A/N247D/Q302K/A337P/A350G/E367D/
324







T369D


284
+++
+++
+++
+++
L44R/K206A/R221T/N247D/M322I/K343D/F365L/
325







I376V


285
+
+
++
+
L44R/K96I/K206A
326


286
+
+
++
+
L44R/K96I/K206A/F217R/N247D
327


287
+++
+++
+++
++
L44R/K96I/K206A/F217R/N247D/Q302K/A337P/
328







A350G


288
+++
+++
+++
+++
L44R/K96I/K206A/F217R/N247D/Q302K/A337P/
329







K343D/A350G/E367D/T369D


289
+
++
++
++
L44R/K96I/K206A/F217R/Q302K/A350G
330


290
+++
+++
+++
+++
L44R/K96I/K206A/N247D/L316D/A337P/A350G/
331







E367D/T369D


291
+
+
+
+
L44R/L100F/K206A/F365L
332


292
+++
++
+++
++
L44R/L100F/K206A/I208V/Q219H/N247D/Q302K/
333







M322V/K343D/R373K/I376V


293
++
+
++
+
L44R/L100F/K206A/I208V/R221K/N247D/Q302K/
334







M322V/F365L/I376V


294
++
++
+++
+
L44R/L100F/K206A/I208V/R221T/N247D/M322V/
335







I376V


295
+++
+++
+++
+++
L44R/L100F/K206A/I208V/R221T/N247D/Q302K/
336







M322I/K343D/F365L/R373K/I376V






1Relative activity was calculated as activity of the variant/activity of Rd2BB (SEQ ID NO: 10 (encoded by SEQ ID NO: 8).




2− = <0.5 relative activity to Rd2BB (SEQ ID NO: 10); + = 0.5 to 1.5 relative activity over Rd2BB (SEQ ID NO: 10); ++ = >1.5 to 2.5 relative activity over Rd2BB (SEQ ID NO: 10); and +++ = >2.5 relative activity over Rd2BB (SEQ ID NO: 10).














TABLE 2.5







Relative Activity of GLA Variants After No Challenge (NC)


or Challenge at the Indicated pH or Condition 1, 2, 3













Variant

pH
pH

Amino Acid Differences Relative to SEQ ID NO: 5
SEQ


#
NC
4.0
8.2
Serum
(WT GLA)
ID NO:
















296
+


+
A66T/K206A/F2I7R/L316D/M322I/A337P/K343G/
337







A350G/E367N/R373K


297




K206A/F217R/G230V/N247D/Q302K/M322I/E367N/
338







T369S/R373K


298
++
+++
+++
+++
K206A/F217R/N247D/L316D/M322I/A337P/A350G/
339







K362Q/E367N/R373K


299
+



K206A/F217R/N247D/Q249H/Q302K/M322I/K343G/
340







A350G/E367T/R373K/L397F


300
+
++
++
++
K206A/I208V/R221T/N247D/M322V/K343G/E367N/
341







R373K


301
+
+
+

K206A/M322I/E367N/R373K
342


302
+
+
+
+
K206A/M322V/K343G/E367N/R373K
343


303
++
++
++
+
K206A/N247D/M322I/A337E/K343D/F365L/E367N/
344







R373K/I376V


304
++
++
++
++
K206A/Q302K/L316D/M322I/A337P/A350G/K362Q/
345







E367N/T369S/R373K


305
+
+
+
++
K206A/Q302K/L316D/M322I/A337P/K343D/E367N/
346







T369S/R373K


306
+
++
++
++
K206A/R221K/N247D/Q302K/M322I/E367N/R373K
347


307
+
+
+
+
K206A/R221K/Q302K/M322I/K343G/E367N/R373K/
348







I376V


308



+
K96I/K206A/F217R/M322I/E367N/T369S/R373K
349


309
+
++
+++
++
K96I/K206A/F217R/N247D/Q302K/M322I/A337P/
350







K343G/A350G/E367N/R373K


310

+
+
+
K96I/K206A/N247D/M322I/A350G/E367N/T369S/
351







R373K


311
+
+
++
+
K96I/K206A/N247D/Q302K/L316D/M322I/A337P/
352







A350G/E367N/T369S/R373K


312
++
+++
+++
+++
K96I/K206A/N247D/Q302K/L316D/M322I/A337P/
353







A350G/K362Q/E367N/T369S/R373K


313
+
+

+
L100F/A125S/K206A/I208V/R221K/Q302K/M322I/
354







K343G/E367N/R373K


314
+
++
++
+
L100F/K206A/I208V/N247D/Q302K/M322V/K343D/
355







E367N/R373K/I376V


315
+
+
+
+
L100F/K206A/I208V/Q302K/M322V/F365L/E367N/
356







R373K/I376V


316
+
+
+
+
L100F/K206A/I208V/R221K/M322V/K343D/E367N/
357







R373K


317
+
+


L100F/K206A/I208V/R221K/M322V/K343D/F365L/
358







E367N/R373K


318
+
+
++
+
L100F/K206A/I208V/R221T/M322V/E367N/R373K/
359







I376V


319
+
+
+
+
L100F/K206A/M322I/E367N/R373K/I376V
360


320
+
+
+
+
L100F/K206A/N247D/Q302K/M322I/E367N/R373K
361


321
++
++
+++
+
L100F/K206A/R221K/N247D/M322I/K343G/E367N/
362







R373K


322
+
+
++
+
L100F/K206A/R221T/Q302K/M322I/K343D/E367N/
363







R373K


323



+
L100F/L160I/K206A/R221K/M322V/E367N/R373K
364


324




L23S/K206A/M322I/E367N/R373K
365


325
++
+++
+++
+++
L44R/K206A/F217R/N247D/L316D/M322I/A337P/
366







K343G/K362Q/E367N/R373K


326
++
+++
+++
+++
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
367







A337P/K362Q/E367N/R373K


327
++
+++
+++
+++
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
368







K343D/A350G/K362Q/E367N/R373K


328
+
+
+
+
L44R/K206A/F217R/Q302K/M322I/A337P/A350G/
369







E367N/T369S/R373K


329
+
++
++
++
L44R/K206A/I208V/N247D/Q302K/M322I/K343D/
370







E367N/R373K


330
++
++
+
+
L44R/K206A/I208V/R221K/M322I/K343D/E367N/
371







R373K


331
+
++
++
+
L44R/K206A/I208V/R221K/N247D/Q302K/M322I/
372







K343D/E367N/R373K/I376V


332
+
+
+
+
L44R/K206A/I208V/R221T/Q302K/M322I/K343G/
373







F365L/E367N/R373K/I376V


333
++
++
++
+
L44R/K206A/L316D/M322I/A337P/A350G/E367N/
374







T369S/R373K


334
+
++
+++
++
L44R/K206A/N247D/L316D/M322I/A350G/K362Q/
375







E367N/T369S/R373K


335
++
+++
+++
+++
L44R/K206A/N247D/Q302K/L316D/M322I/A337P/
376







K343G/A350G/K362Q/E367N/T369S/R373K


336
+
+
+

L44R/K206A/N247D/Q302K/M322I/A350G/E367N/
377







T369S/R373K


337
+
++
++
++
L44R/K206A/N247D/Q302K/M322I/K343D/E367N/
378







R373K


338
++
+++
+++
+++
L44R/K96I/K206A/F217R/N247D/L316D/M322I/
379







A337P/A350G/K362Q/E367N/R373K


339
+
+
++
+
L44R/K96I/K206A/F217R/N247D/M322I/A350G/
380







K362Q/E367N/R373K


340
+
+
+
+
L44R/K96I/K206A/F217R/N247D/M322I/A350G/
381







K362Q/E367N/T369S/R373K


341

+
+
+
L44R/K96I/K206A/F217R/N247D/M322I/E367N/
382







T369S/R373K


342
++
+++
+++
+++
L44R/K96I/K206A/F217R/N247D/Q302K/L316D/
383







M322I/A337P/E367N/R373K


343
+
+
+
+
L44R/K96I/K206A/F217R/N247D/Q302K/M322I/
384







E367N/T369S/R373K


344
+
+
+
++
L44R/K96I/K206A/F217R/N247D/Q302K/M322I/
385







K362Q/E367N/R373K


345
+
++
+++
+
L44R/K96I/K206A/F217R/Q219P/N247D/M253K/
386







S266F/D284E/Q290P/L293F/Q302K/V308G/S314F/







M322I/A337P/K343E/E367N/R373K


346
+
+
++
+
L44R/K96I/K206A/F217R/Q302K/M322I/A350G/
387







K362Q/E367N/T369S/R373K


347




L44R/K96I/K206A/M322I/A337P/E367N/T369S/
388







R373K


348
+
+
+
+
L44R/L100F/K206A/I208V/R221K/M322I/K343G/
389







F365L/E367N/R373K


349
+
+
++
+
L44R/L100F/K206A/I208V/R221T/N247D/M322I/
390







F365L/E367N/R373K


350
+
++
+++
+
L44R/L100F/K206A/I208V/R221T/N247D/M322V/
391







E367N/R373K/I376V


351
+
+
++
+
L44R/L100F/K206A/I208V/R221T/Q302K/M322I/
392







E367N/R373K/I376V


352
+
+
+
+
L44R/L100F/K206A/Q302K/M322I/E367N/R373K/
393







I376V


353



+
L44R/L100F/K206A/R221K/M322I/F365L/E367N/
394







R373K/I376V


354

+
+
+
L44R/L100F/K206A/R221T/M322I/F365L/E367N/
395







R373K


355
+
++
++
+
L44R/L100F/K206A/R221T/N247D/M322I/K343D/
396







E367N/R373K/I376V


356
+
+
++
+
L44R/L100F/K206A/R221T/N247D/Q302K/M322I/
397







E367N/R373K


357
+
++
+++
+
L44R/L100F/K206A/R221T/N247D/Q302K/M322V/
398







E367N/R373K/I376V


358
+
+
++
+
L44R/L100F/K206A/R221T/Q302K/M322I/E367N/
399







R373K


359
+
+
+++
+
L44R/L100F/Q181L/K206A/R221T/N247D/Q302K/
400







M322V/E367N/R373K/I376V






1 Relative activity was calculated as activity of the variant/activity of Rd3BB (SEQ ID NO: 13 (encoded by SEQ ID NO: 11).




2 Variant # 326 (Rd4BB) has the polynucleotide sequence of SEQ ID NO: 14 and polypeptide sequence of SEQ ID NO: 15.




3 − = <0.5 relative activity to Rd3BB (SEQ ID NO: 13); + = 0.5 to 1.5 relative activity over Rd3BB (SEQ ID NO: 13); ++ = >1.5 to 2.5 relative activity over Rd3BB (SEQ ID NO: 13); and +++ = >2.5 relative activity over Rd3BB (SEQ ID NO: 13).














TABLE 2.6







Relative Activity of GLA Variants After No Challenge (NC)


or Challenge at the Indicated pH or Condition 1, 2, 3, 4












Variant

pH
pH
Amino acid differences relative to SEQ ID NO: 5
SEQ


#
NC
3.7
9.65
(WT GLA)
ID NO:















360
+
+
+
L44E/K206A/F217R/N247D/Q302K/L316D/M322I/
401






A337P/K362Q/E367N/R373K


361
+
+
+
L44R/S47R/K206A/F217R/N247D/Q302K/L316D/
402






M322I/A337P/K362Q/E367N/R373K


362
+
+
+
L44C/K206A/F217R/N247D/Q302K/L316D/M322I/
403






A337P/K362Q/E367N/R373K


363
+
+
+
L44R/S47D/K206A/F217R/N247D/Q302K/L316D/
404






M322I/A337P/K362Q/E367N/R373K


364
+
+

M39H/L44R/K206A/F217R/N247D/Q302K/L316D/
405






M322I/A337P/K362Q/E367N/R373K


365
+
+
+
L44R/S47N/K206A/F217R/N247D/Q302K/L316D/
406






M322I/A337P/K362Q/E367N/R373K


366
+
+
+
L44R/S47V/K206A/F217R/N247D/Q302K/L316D/
407






M322I/A337P/K362Q/E367N/R373K


367
+
+

M39R/L44R/K206A/F217R/N247D/Q302K/L316D/
408






M322I/A337P/K362Q/E367N/R373K


368
+
+
+
L44A/K206A/F217R/N247D/Q302K/L316D/M322I/
409






A337P/K362Q/E367N/R373K


369
+
+

L44S/K206A/F217R/N247D/Q302K/L316D/M322I/
410






A337P/K362Q/E367N/R373K


370
+
++
+
L44Q/K206A/F217R/N247D/Q302K/L316D/M322I/
411






A337P/K362Q/E367N/R373K


371
+

+
L44W/K206A/F217R/N247D/Q302K/L316D/M322I/
412






A337P/K362Q/E367N/R373K


372
+

+
L44V/K206A/F217R/N247D/Q302K/L316D/M322I/
413






A337P/K362Q/E367N/R373K


373



M41R/L44R/K206A/F217R/N247D/Q302K/L316D/
414






M322I/A337P/K362Q/E367N/R373K


374
+
+
+
L44M/K206A/F217R/N247D/Q302K/L316D/M322I/
415






A337P/K362Q/E367N/R373K


375
+
+
+
L44R/S47I/K206A/F217R/N247D/Q302K/L316D/
416






M322I/A337P/K362Q/E367N/R373K


376



M41P/L44R/K206A/F217R/N247D/Q302K/L316D/
417






M322I/A337P/K362Q/E367N/R373K


377
+
++

M39T/L44R/K206A/F217R/N247D/Q302K/L316D/
418






M322I/A337P/K362Q/E367N/R373K


378
+

+
L44T/K206A/F217R/N247D/Q302K/L316D/M322I/
419






A337P/K362Q/E367N/R373K


379
+
++
+
L44R/S47T/K206A/F217R/N247D/Q302K/L316D/
420






M322I/A337P/K362Q/E367N/R373K


380
+
+

L44R/Y92K/K206A/F217R/N247D/Q302K/L316D/
421






M322I/A337P/K362Q/E367N/R373K


381
+
+

L44R/Y92S/K206A/F217R/N247D/Q302K/L316D/
422






M322I/A337P/K362Q/E367N/R373K


382



L44R/H94N/K206A/F217R/N247D/Q302K/L316D/
423






M322I/A337P/K362Q/E367N/R373K


383
+


L44R/Y92C/K206A/F217R/N247D/Q302K/L316D/
424






M322I/A337P/K362Q/E367N/R373K


384
+
+

L44R/Y92V/K206A/F217R/N247D/Q302K/L316D/
425






M322I/A337P/K362Q/E367N/R373K


385
+
+

L44R/Y92A/K206A/F217R/N247D/Q302K/L316D/
426






M322I/A337P/K362Q/E367N/R373K


386

+

L44R/H94R/K206A/F217R/N247D/Q302K/L316D/
427






M322I/A337P/K362Q/E367N/R373K


387
+
+

L44R/V93T/K206A/F217R/N247D/Q302K/L316D/
428






M322I/A337P/K362Q/E367N/R373K


388
+

+
L44R/V93L/K206A/F217R/N247D/Q302K/L316D/
429






M322I/A337P/K362Q/E367N/R373K


389
+
+

L44R/V93S/K206A/F217R/N247D/Q302K/L316D/
430






M322I/A337P/K362Q/E367N/R373K


390
+
+

L44R/Y92Q/K206A/F217R/N247D/Q302K/L316D/
431






M322I/A337P/K362Q/E367N/R373K


391
+
+

L44R/Y92W/K206A/F217R/N247D/Q302K/L316D/
432






M322I/A337P/K362Q/E367N/R373K/L397S


392
+
+

L44R/Y92T/K206A/F217R/N247D/Q302K/L316D/
433






M322I/A337P/K362Q/E367N/R373K


393
+


L44R/Y92G/K206A/F217R/N247D/Q302K/L316D/
434






M322I/A337P/K362Q/E367N/R373K


394
+
+

L44R/Y92R/K206A/F217R/N247D/Q302K/L316D/
435






M322I/A337P/K362Q/E367N/R373K


395
+
+
+
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
40






M322I/A337P/K362Q/E367N/R373K


396
+
+
+
L44R/L158M/K206A/F217R/N247D/Q302K/L316D/
437






M322I/A337P/K362Q/E367N/R373K


397
+
+
+
L44R/L158R/K206A/F217R/N247D/Q302K/L316D/
438






M322I/A337P/K362Q/E367N/R373K


398
+
++

L44R/A159S/K206A/F217R/N247D/Q302K/L316D/
439






M322I/A337P/K362Q/E367N/R373K


399
+
+
+
L44R/R165K/K206A/F217R/N247D/Q302K/L316D/
440






M322I/A337P/K362Q/E367N/R373K


400
+
+

L44R/L158C/K206A/F217R/N247D/Q302K/L316D/
441






M322I/A337P/K362Q/E367N/R373K


401
+
+

L44R/T163S/K206A/F217R/N247D/Q302K/L316D/
442






M322I/A337P/K362Q/E367N/R373K


402
+
++
+
L44R/S166P/K206A/F217R/N247D/Q302K/L316D/
42






M322I/A337P/K362Q/E367N/R373K


403
+
+
+
L44R/S166G/K206A/F217R/N247D/Q302K/L316D/
444






M322I/A337P/K362Q/E367N/R373K


404
+
+

L44R/S166F/K206A/F217R/N247D/Q302K/L316D/
445






M322I/A337P/K362Q/E367N/R373K


405
+
++
+
L44R/L158E/K206A/F217R/N247D/Q302K/L316D/
446






M322I/A337P/K362Q/E367N/R373K


406
+
+
+
L44R/R162K/K206A/F217R/N247D/Q302K/L316D/
447






M322I/A337P/K362Q/E367N/R373K


407
+
+

L44R/L158H/K206A/F217R/N247D/Q302K/L316D/
448






M322I/A337P/K362Q/E367N/R373K


408
+
+
+
L44R/S166R/K206A/F217R/N247D/Q302K/L316D/
449






M322I/A337P/K362Q/E367N/R373K


409
+
+

L44R/R165H/K206A/F217R/N247D/Q302K/L316D/
450






M322I/A337P/K362Q/E367N/R373K


410
+
+

L44R/R162H/K206A/F217R/N247D/Q302K/L316D/
451






M322I/A337P/K362Q/E367N/R373K


411
+
+
+
L44R/S166A/K206A/F217R/N247D/Q302K/L316D/
452






M322I/A337P/K362Q/E367N/R373K


412
+
++
+
L44R/S166H/K206A/F217R/N247D/Q302K/L316D/
453






M322I/A337P/K362Q/E367N/R373K


413



L44R/T163*/K206A/F217R/N247D/Q302K/L316D/
454






M322I/A337P/K362Q/E367N/R373K


414
+
+
+
L44R/L158Q/K206A/F217R/N247D/Q302K/L316D/
455






M322I/A337P/K362Q/E367N/R373K


415
+
+
+
L44R/S166D/K206A/F217R/N247D/Q302K/L316D/
456






M322I/A337P/K362Q/E367N/R373K


416
+
+

L44R/R162G/K206A/F217R/N247D/Q302K/L316D/
457






M322I/A337P/K362Q/E367N/R373K


417
+
+

L44R/R162S/K206A/F217R/N247D/Q302K/L316D/
458






M322I/A337P/K362Q/E367N/R373K


418
+
+

L44R/N161E/K206A/F217R/N247D/Q302K/L316D/
459






M322I/A337P/K362Q/E367N/R373K


419
+
+
+
L44R/S166E/K206A/F217R/N247D/Q302K/L316D/
460






M322I/A337P/K362Q/E367N/R373K


420
+
++

L44R/S166T/K206A/F217R/N247D/Q302K/L316D/
461






M322I/A337P/K362Q/E367N/R373K


421
+
+

L44R/R162Q/K206A/F217R/N247D/Q302K/L316D/
462






M322I/A337P/K362Q/E367N/R373K


422
+
+

L44R/L158G/K206A/F217R/N247D/Q302K/L316D/
463






M322I/A337P/K362Q/E367N/R373K


423
+
+
+
L44R/R162A/K206A/F217R/N247D/Q302K/L316D/
464






M322I/A337P/K362Q/E367N/R373K


424
+
+

L44R/K206A/F217R/N247D/L255E/Q302K/L316D/
465






M322I/A337P/K362Q/E367N/R373K


425
+

+
L44R/K206A/F217R/N247D/H271E/Q302K/L316D/
466






M322I/A337P/K362Q/E367N/R373K


426
+


L44R/K206A/F217R/N247D/M259E/Q302K/L316D/
467






M322I/A337P/K362Q/E367N/R373K


427
+


L44R/K206A/F217R/N247D/L263G/Q302K/L316D/
468






M322I/A337P/K362Q/E367N/R373K


428
+
+

L44R/K206A/F217R/N247D/M259S/Q302K/L316D/
469






M322I/A337P/K362Q/E367N/R373K


429
+
+

L44R/K206A/F217R/N247D/L255C/Q302K/L316D/
470






M322I/A337P/K362Q/E367N/R373K


430
+

+
L44R/K206A/F217R/N247D/H271T/Q302K/L316D/
471






M322I/A337P/K362Q/E367N/R373K


431
+


L44R/K206A/F217R/N247D/R270G/Q302K/L316D/
472






M322I/A337P/K362Q/E367N/R373K


432
+

+
L44R/K206A/F217R/N247D/L255V/Q302K/L316D/
473






M322I/A337P/K362Q/E367N/R373K


433
+
+
+
L44R/K206A/F217R/N247D/H271Q/Q302K/L316D/
474






M322I/A337P/K362Q/E367N/R373K


434
+


L44R/K206A/F217R/N247D/R270D/Q302K/L316D/
475






M322I/A337P/K362Q/E367N/R373K


435
+
++

L44R/K206A/F217R/N247D/I258L/Q302K/L316D/
476






M322I/A337P/K362Q/E367N/R373K


436
+


L44R/K206A/F217R/N247D/H271G/Q302K/L316D/
477






M322I/A337P/K362Q/E367N/R373K


437
+
+

L44R/K206A/F217R/N247D/L263E/Q302K/L316D/
478






M322I/A337P/K362Q/E367N/R373K


438



L44R/K206A/F217R/N247D/L255*/Q302K/L316D/
479






M322I/A337P/K362Q/E367N/R373K


439
+
+
+
L44R/K206A/F217R/N247D/H271A/Q302K/L316D/
480






M322I/A337P/K362Q/E367N/R373K


440
+
+

L44R/K206A/F217R/N247D/L263C/Q302K/L316D/
481






M322I/A337P/K362Q/E367N/R373K


441
+

+
L44R/K206A/F217R/N247D/H271V/Q302K/L316D/
482






M322I/A337P/K362Q/E367N/R373K


442
+
+

L44R/K206A/F217R/N247D/L255A/Q302K/L316D/
483






M322I/A337P/K362Q/E367N/R373K


443
+
+++

L44R/K206A/F217R/N247D/L255S/Q302K/L316D/
484






M322I/A337P/K362Q/E367N/R373K


444
+

+
L44R/K206A/F217R/N247D/M259W/Q302K/L316D/
485






M322I/A337P/K362Q/E367N/R373K


445
+
+

L44R/K206A/F217R/N247D/L263F/Q302K/L316D/
486






M322I/A337P/K362Q/E367N/R373K


446
+


L44R/K206A/F217R/N247D/M259A/Q302K/L316D/
487






M322I/A337P/K362Q/E367N/R373K


447
+


L44R/K206A/F217R/N247D/L263W/Q302K/L316D/
488






M322I/A337P/K362Q/E367N/R373K


448
+


L44R/K206A/F217R/N247D/R270Q/Q302K/L316D/
489






M322I/A337P/K362Q/E367N/R373K


449
+
++

L44R/K206A/F217R/N247D/L255I7Q302K/L316D/
490






M322I/A337P/K362Q/E367N/R373K


450
+
++

L44R/K206A/F217R/N247D/I258M/Q302K/L316D/
491






M322I/A337P/K362Q/E367N/R373K


451
+
++

L44R/K206A/F217R/N247D/M259V/Q302K/L316D/
492






M322I/A337P/K362Q/E367N/R373K


452
+
++
+
L44R/K206A/F217R/N247D/H271R/Q302K/L316D/
493






M322I/A337P/K362Q/E367N/R373K


453
+


L44R/K206A/F217R/N247D/R270L/Q302K/L316D/
494






M322I/A337P/K362Q/E367N/R373K


454
+
++
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
495






A337P/K362Q/E367N/R373K/M390P


455
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
496






A337P/K362Q/E367N/R373K/M392D


456
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
497






A337P/K362Q/E367N/R373K/T389M


457
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
498






A337P/K362Q/E367N/R373K/M392A


458
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
499






A337P/K362Q/E367N/R373K/M390*


459
+
++
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
500






A337P/K362Q/E367N/R373K/M390H


460
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
501






A337P/K362Q/E367N/R373K/L386T


461
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
502






A337P/K362Q/E367N/R373K/M392Q


462
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
503






A337P/K362Q/E367N/R373K/Q385L


463
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
504






A337P/K362Q/E367N/R373K/M390T


464
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
505






A337P/K362Q/E367N/R373K/M392*


465
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
506






A337P/K362Q/E367N/R373K/M390Q


466
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
507






A337P/K362Q/E367N/R373K/M392E


467
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
508






A337P/K362Q/E367N/R373K/T389S


468
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
509






A337P/K362Q/E367N/R373K/T389Q


469
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
510






A337P/K362Q/E367N/R373K/Q385I


470
+
++
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
511






A337P/K362Q/E367N/R373K/M392R


471
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
512






A337P/K362Q/E367N/R373K/T389W


472
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
513






A337P/K362Q/E367N/R373K/M392K


473
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
514






A337P/K362Q/E367N/R373K/M392L


474
+
++
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
515






A337P/K362Q/E367N/R373K/L386F


475
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
516






A337P/K362Q/E367N/R373K/T389D


476
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
517






A337P/K362Q/E367N/R373K/M390E


477
+

+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
518






A337P/K362Q/E367N/R373K/L384W


478
+
++
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
519






A337P/K362Q/E367N/R373K/M392S


479
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
520






A337P/K362Q/E367N/R373K/M392F


480
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
521






A337P/K362Q/E367N/R373K/M390R


481
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
522






A337P/K362Q/E367N/R373K/M390G


482
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
523






A337P/K362Q/E367N/R373K/Q385G


483
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
524






A337P/K362Q/E367N/R373K/M392C


484
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
525






A337P/K362Q/E367N/R373K/M392V


485
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
526






A337P/K362Q/E367N/R373K/M392W


486
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
527






A337P/K362Q/E367N/R373K/M390C


487
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
528






A337P/K362Q/E367N/R373K/T389G


488
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
529






A337P/K362Q/E367N/R373K/T389N


489
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
530






A337P/K362Q/E367N/R373K/T389I


490
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
531






A337P/K362Q/E367N/R373K/M390D


491
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
532






A337P/K362Q/E367N/R373K/M390W


492
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
533






A337P/K362Q/E367N/R373K/T389C


493
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
534






A337P/K362Q/E367N/R373K/M392P


494
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
535






A337P/K362Q/E367N/R373K/M390F


495
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
536






A337P/K362Q/E367N/R373K/T389P


496
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
537






A337P/K362Q/E367N/R373K/M390V


497
+
++
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
538






A337P/K362Q/E367N/R373K/M390K


498
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
539






A337P/K362Q/E367N/R373K/M392I


499
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
540






A337P/K362Q/E367N/R373K/T389L


500
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
541






A337P/K362Q/E367N/R373K/M390A


501
+
++
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
542






A337P/K362Q/E367N/R373K/M392G


502

+

L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
543






A337P/K362Q/E367N/R373K/L386S


503
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
544






A337P/K362Q/E367N/R373K/Q385C


504
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
545






A337P/K362Q/E367N/R373K/M390S


505
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
546






A337P/K362Q/E367N/R373K/M392N


506
+
+

L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
547






A337P/K362Q/E367N/R373K/Q385W


507
+
++
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
548






A337P/K362Q/E367N/R373K/M392T


508



L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
549






A337P/K362Q/E367N/R373K/L384A


509
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
550






A337P/K362Q/E367N/R373K/Q385T


510
+

+
L44R/A199G/K206A/F217R/N247D/Q302K/L316D/
551






M322I/A337P/K362Q/E367N/R373K/M392R


511
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
552






A337P/K362Q/E367N/R373K/L397*


512
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
553






A337P/K362Q/E367N/R373K/K395*


513
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
554






A337P/K362Q/E367N/R373K/D396*


514
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
555






A337P/K362Q/E367N/R373K/S393*


515
+
+
+
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/
556






A337P/K362Q/E367N/R373K/L394*






1 Relative activity was calculated as activity of the variant/activity of Rd4BB (SEQ ID NO: 15 (encoded by SEQ ID NO: 14).




2 Variant # 395 (Rd5BB) has the polynucleotide sequence of SEQ ID NO: 39 and polypeptide sequence of SEQ ID NO: 40.




3 Variant # 402 (Rd6BB) has the polynucleotide sequence of SEQ ID NO: 41 and polypeptide sequence of SEQ ID NO: 42




4 − = <0.5 relative activity to Rd4BB (SEQ ID NO: 15); + = 0.5 to 1.5 relative activity over Rd4BB (SEQ ID NO: 15); ++ = >1.5 to 2.5 relative activity over Rd4BB (SEQ ID NO: 15); and +++ = >2.5 relative activity over Rd4BB (SEQ ID NO: 15).














TABLE 2.7







Relative Activity of GLA Variants After No Challenge


(NC) or Challenge at the Indicated pH or Condition












Variant

pH
pH
Amino acid differences relative to SEQ ID NO: 5
SEQ


#
NC
3.7
9.7
(WT GLA)
ID NO:















516
+
+++
+
D2E/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
557






M322I/Q326G/A337P/K362Q/E367N/R373K


517
+
+
+
D2Q/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
558






M322I/A337P/K362Q/E367N/R373K


518
+
+
++
E40D/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
559






M322I/A337P/K362Q/E367N/R373K


519
+
+
++
E40S/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
560






M322I/A337P/K362Q/E367N/R373K


520
+
+
+
L44R/A77S/Y92H/K206A/F217R/N247D/Q302K/L316D/
561






M322I/A337P/K362Q/E367N/R373K


521
+
+
++
L44R/D52N/Y92H/K206A/F217R/N247D/Q302K/L316D/
562






M322I/A337P/K362Q/E367N/R373K


522
+
+++
++
L44R/E56K/Y92H/K206A/F217R/N247D/Q302K/L316D/
563






M322I/A337P/K362Q/E367N/R373K


523
+
+
+
L44R/N91M/Y92H/K206A/F217R/N247D/Q302K/L316D/
564






M322I/A337P/K362Q/E367N/R373K


524

+
+
L44R/N91V/Y92H/K206A/F217R/N247D/Q302K/L316D/
565






M322I/A337P/K362Q/E367N/R373K


525
+
++
++
L44R/Q76H/Y92H/K206A/F217R/N247D/Q302K/L316D/
566






M322I/A337P/K362Q/E367N/W368A/R373K


526
+
+
++
L44R/R74H/Y92H/K206A/F217R/N247D/Q302K/L316D/
567






M322I/A337P/K362Q/E367N/R373K


527
+
+
++
L44R/Y92E/K206A/F217R/N247D/Q302K/L316D/M322I/
568






A337P/K362Q/E367N/R373K


528
+
+
++
L44R/Y92H/D130Q/K206A/F217R/N247D/Q302K/L316D/
569






M322I/A337P/K362Q/E367N/R373K


529
+
+
++
L44R/Y92H/K182A/K206A/F217R/N247D/Q302K/L316D/
570






M322I/A337P/K362Q/E367N/R373K


530
+
+
++
L44R/Y92H/K182E/K206A/F217R/N247D/Q302K/L316D/
571






M322I/A337P/K362Q/E367N/R373K


531
+
+
++
L44R/Y92H/K182H/K206A/F217R/N247D/Q302K/L316D/
572






M322I/A337P/K362Q/E367N/R373K


532
+
+
++
L44R/Y92H/K182M/K206A/F217R/N247D/Q302K/L316D/
573






M322I/A337P/K362Q/E367N/R373K


533
+
+
++
L44R/Y92H/K182Q/K206A/F217R/N247D/Q302K/L316D/
574






M322I/A337P/K362Q/E367N/R373K


534
+
+
++
L44R/Y92H/K182R/K206A/F217R/N247D/Q302K/L316D/
575






M322I/A337P/K362Q/E367N/R373K


535
+
+
++
L44R/Y92H/K182T/K206A/F217R/N247D/Q302K/L316D/
576






M322I/A337P/K362Q/E367N/R373K


536
+
+
++
L44R/Y92H/K182V/K206A/F217R/N247D/Q302K/L316D/
577






M322I/A337P/K362Q/E367N/R373K


537
+
+
++
L44R/Y92H/K182Y/K206A/F217R/N247D/Q302K/L316D/
578






M322I/A337P/K362Q/E367N/R373K


538
+
+
+
L44R/Y92H/K206A/F217R/N247D/A287C/Q302K/L316D/
579






M322I/A337P/K362Q/E367N/R373K


539
+
+
++
L44R/Y92H/K206A/F217R/N247D/A287H/Q302K/L316D/
580






M322I/A337P/K362Q/E367N/R373K


540
+
+
++
L44R/Y92H/K206A/F217R/N247D/A287M/Q302K/L316D/
581






M322I/A337P/K362Q/E367N/R373K


541
+
+
++
L44R/Y92H/K206A/F217R/N247D/K283A/Q302K/L316D/
582






M322I/A337P/K362Q/E367N/R373K


542
+
+
++
L44R/Y92H/K206A/F217R/N247D/K283G/Q302K/L316D/
583






M322I/A337P/K362Q/E367N/R373K


543
+
+
+
L44R/Y92H/K206A/F217R/N247D/K283M/Q302K/L316D/
584






M322I/A337P/K362Q/E367N/R373K


544
+
+
+
L44R/Y92H/K206A/F217R/N247D/K283V/Q302K/L316D/
585






M322I/A337P/K362Q/E367N/R373K


545
+
+
++
L44R/Y92H/K206A/F217R/N247D/K295A/Q302K/L316D/
586






M322I/A337P/K362Q/E367N/R373K


546
+
+++
++
L44R/Y92H/K206A/F217R/N247D/K295E/Q302K/L316D/
587






M322I/A337P/K362Q/E367N/R373K


547
+
+
++
L44R/Y92H/K206A/F217R/N247D/K295L/Q302K/L316D/
588






M322I/A337P/K362Q/E367N/R373K


548
+
+++
++
L44R/Y92H/K206A/F217R/N247D/K295N/Q302K/L316D/
589






M322I/A337P/K362Q/E367N/R373K


549
+
++
++
L44R/Y92H/K206A/F217R/N247D/K295Q/Q302K/L316D/
590






M322I/A337P/K362Q/E367N/R373K


550
+
+
++
L44R/Y92H/K206A/F217R/N247D/K295S/Q302K/L316D/
591






M322I/A337P/K362Q/E367N/R373K


551
+
+
++
L44R/Y92H/K206A/F217R/N247D/K295T/Q302K/L316D/
592






M322I/A337P/K362Q/E367N/R373K


552
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/A317D/
593






M322I/A337P/K362Q/E367N/R373K


553
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/A317Q/
594






M322I/A337P/K362Q/E367N/R373K


554
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
595






A337P/A346G/K362Q/E367N/R373K


555
+
+
+
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
596






A337P/G344A/K362Q/E367N/R373K


556


+
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
597






A337P/G344D/K362Q/E367N/R373K


557
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
598






A337P/G344S/K362Q/E367N/R373K


558


+
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
599






A337P/I353L/K362Q/E367N/R373K


559
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
600






A337P/K362Q/E367N/L372W/R373K


560
+
++
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
601






A337P/K362Q/E367N/W368A/R373K


561
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
602






A337P/K362Q/E367N/W368L/R373K


562
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
603






A337P/K362Q/E367N/W368N/R373K


563
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
604






A337P/K362Q/E367N/W368R/R373K


564
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
605






A337P/K362Q/E367N/W368V/R373K


565
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
606






A337P/N348E/K362Q/E367N/R373K


566
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
607






A337P/N348M/K362Q/E367N/R373K


567
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
608






A337P/N348Q/K362Q/E367N/R373K


568
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
609






A337P/N348R/K362Q/E367N/R373K


569
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
610






A337P/N348W/K362Q/E367N/R373K


570
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
611






A337P/T354S/K362Q/E367N/R373K


571
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/N305K/L316D/
612






M322I/A337P/K362Q/E367N/R373K


572
+
+++
++
L44R/Y92H/K206A/F217R/N247D/Q302K/N305L/L316D/
613






M322I/A337P/K362Q/E367N/R373K


573
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/S314A/L316D/
614






M322I/A337P/K362Q/E367N/R373K


574
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/S314H/L316D/
615






M322I/A337P/K362Q/E367N/R373K


575
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/S314N/L316D/
616






M322I/A337P/K362Q/E367N/R373K


576
+
+
++
L44R/Y92H/K206A/F217R/N247D/Q302K/S314Y/L316D/
617






M322I/A337P/K362Q/E367N/R373K


577
+
+++
++
L44R/Y92H/K206A/F217R/W246A/N247D/Q302K/L316D/
618






M322I/A337P/K362Q/E367N/R373K


578
+
+++
++
L44R/Y92H/K206A/F217R/W246I/N247D/Q302K/L316D/
619






M322I/A337P/K362Q/E367N/R373K


579
+
+++
++
L44R/Y92H/K206A/F217R/W246P/N247D/Q302K/L316D/
620






M322I/A337P/K362Q/E367N/R373K


580
+
+++
++
L44R/Y92H/K206A/F217R/W246R/N247D/Q302K/L316D/
621






M322I/A337P/K362Q/E367N/R373K


581
+
++
++
L44R/Y92H/K206A/F217R/W246S/N247D/Q302K/L316D/
622






M322I/A337P/K362Q/E367N/R373K


582
+
+
++
L44R/Y92H/K206A/S210A/F217R/N247D/Q302K/L316D/
623






M322I/A337P/A350T/K362Q/E367N/R373K


583
+
+
++
L44R/Y92H/K206A/S210A/F217R/N247D/Q302K/L316D/
624






M322I/A337P/K362Q/E367N/R373K


584
+
+
++
L44R/Y92H/K206A/S210E/F217R/N247D/Q302K/L316D/
625






M322I/A337P/K362Q/E367N/R373K


585
+
+
++
L44R/Y92H/K206A/S210K/F217R/N247D/Q302K/L316D/
626






M322I/A337P/K362Q/E367N/R373K


586
+
++
++
L44R/Y92H/K206A/S210N/F217R/N247D/Q302K/L316D/
627






M322I/A337P/K362Q/E367N/R373K


587
+
+
++
L44R/Y92H/K206A/S210R/F217R/N247D/Q302K/L316D/
628






M322I/A337P/K362Q/E367N/R373K


588
+
+
+
L44R/Y92H/K96A/K206A/F217R/N247D/Q302K/L316D/
629






M322I/A337P/K362Q/E367N/R373K


589
+
+
+
L44R/Y92H/K96W/K206A/F217R/N247D/Q302K/L316D/
630






M322I/A337P/K362Q/E367N/R373K


590
+
+
+
L44R/Y92H/P179M/K206A/F217R/N247D/Q302K/L316D/
631






M322I/A337P/K362Q/E367N/R373K


591
+
+
++
L44R/Y92H/R189K/K206A/F217R/N247D/Q302K/L316D/
632






M322I/A337P/K362Q/E367N/R373K


592
+
+
++
L44R/Y92H/R189V/K206A/F217R/N247D/Q302K/L316D/
633






M322I/A337P/K362Q/E367N/R373K


593
+
+
++
L44R/Y92H/S95A/K206A/F217R/N247D/Q302K/L316D/
634






M322I/A337P/K362Q/E367N/R373K


594
+
+
++
L44R/Y92H/S95E/K206A/F217R/N247D/Q302K/L316D/
635






M322I/A337P/K362Q/E367N/R373K


595
+
+
+
L44R/Y92H/T186A/K206A/F217R/N247D/Q302K/L316D/
636






M322I/A337P/K362Q/E367N/R373K


596
+
++
++
L44R/Y92H/T186G/K206A/F217R/N247D/Q302K/L316D/
637






M322I/A337P/K362Q/E367N/R373K


597
+

+
L44R/Y92H/T186V/K206A/F217R/N247D/Q302K/L316D/
638






M322I/A337P/K362Q/E367N/R373K


598
+
+
++
L44R/Y92H/Y120H/K206A/F217R/N247D/Q302K/L316D/
639






M322I/A337P/K362Q/E367N/R373K


599
+
+
++
L44R/Y92H/Y120S/K206A/F217R/N247D/Q302K/L316D/
640






M322I/A337P/K362Q/E367N/R373K


600
+
+++
+
L44R/Y92H/Y120S/K206A/F217R/N247D/Q302K/L316D/
641






M322I/A337P/L341F/K362Q/E367N/R373K


601

+
+
M39C/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
642






M322I/A337P/K362Q/E367N/R373K


602
+
+
++
M39E/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
643






M322I/A337P/K362Q/E367N/R373K


603
+
+
+
M39R/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
644






M322I/A337P/K362Q/E367N/R373K


604
+
+
++
M39V/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
645






M322I/A337P/K362Q/E367N/R373K


605
+
+++
++
T10P/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/
646






M322I/A337P/K362Q/E367N/R373K


606
+
+++
++
T10P; L44R; Y92H; R189L; K206A; F217R; N247D;
647






Q302K; L316D; M322I; A337P; K362Q; E367N; R373K


607
+
+
++
T8L; L44R; Y92H; K206A; F217R; N247D; Q302K;
648






L316D; M322I; A337P; K362Q; E367N; R373K


608
+
+
+
T8Q; L44R; Y92H; K206A; F217R; N247D; Q302K;
649






L316D; M322I; A337P; K362Q; E367N; R373K





1. Relative activity was calculated as activity of the variant/activity of Rd3BB (SEQ ID NO: 13) (encoded by SEQ ID NO: 11).


2. − = <1.5 relative activity to Rd3BB (SEQ ID NO: 13); + = 1.5 to 5 relative activity over Rd3BB (SEQ ID NO: 13); ++ = >5 to 10 relative activity over Rd3BB (SEQ ID NO: 13); and +++ = >10 relative activity over Rd3BB (SEQ ID NO: 13).













TABLE 2.8







Relative Activity of GLA Variants After No Challenge


(NC) or Challenge at the Indicated pH or Condition












Variant

pH
pH
Amino acid differences relative to SEQ ID NO: 5
SEQ


#
NC
3.3
9.7
(WT GLA)
ID NO:















609
+
++
++
L44R/S166P/K206A/F217R/N247D/Q302K/L316D/M322I/
650






A337P/K362Q/E367N/R373K


610
+
+

L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M259E/
651






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


611
+
+++
++
L44R/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/
652






M322I/A337P/K362Q/E367N/R373K/M390Q


612
+
++
++
L44R/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/
653






M322I/A337P/K362Q/E367N/R373K/M392T


613
+
++
++
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/H271A/
654






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


614
+
++
++
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/Q302K/
655






L316D/M322I/A337P/K362Q/E367N/R373K


615
+
++
++
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/Q302K/
656






L316D/M3221/A337P/K362Q/E367N/R373K/M390H


616
+
+
++
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M259W/
657






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/






M390Q


617
+
++
++
L44R/Y92H/L136V/S166P/K206A/F217R/N247D/M259A/
658






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


618
+
++
++
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
659






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K


619
+
++
++
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
660






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390H


620

+
++
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
661






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


621
+
++

L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M259E/
662






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/






M390Q


622
+

++
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/M259W/
663






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/






M390Q/M392T


623
+
+++
++
L44R/S47N/S166P/K206A/F217R/N247D/H271A/A276S/
664






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


624
+
++
++
L44R/S47N/S166P/K206A/F217R/N247D/H271A/Q302K/
665






L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


625
+
++
++
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
44






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


626
+
++
++
L44R/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
666






L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


627
+

++
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/M259W/
667






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/






M390H/M392T


628
+
+
++
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/H271A/
668






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390H


629
+
++
++
L44R/S47T/S166P/K206A/F217R/N247D/H271A/Q302K/
669






L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


630
+

++
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M259W/
670






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/






M390H


631
+
+
++
L44R/S47T/A53S/Y92H/S166P/K206A/F217R/N247D/
671






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/






M390Q


632
+
++
++
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/H271A/
672






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


633
+
++
++
E43D/L44R/Y92S/S166P/K206A/F217R/N247D/Q302K/
673






L316D/M322I/A337P/K362Q/E367N/R373K


634
+
++
++
E43D/L44R/Y92E/S166P/K206A/F217R/N247D/Q302K/
674






L316D/M322I/A337P/K362Q/E367N/R373K


635
+
++
++
E43D/L44R/Y92H/S166P/K206A/F217R/N247D/Q302K/
675






L316D/M322I/A337P/K362Q/E367N/R373K


636
+
+
++
E43D/L44R/Y92N/S166P/K206A/F217R/N247D/Q302K/
676






L316D/M322I/A337P/K362Q/E367N/R373K


637
+
+
++
E43Q/L44R/Y92E/S166P/K206A/F217R/N247D/Q302K/
677






L316D/M322I/A337P/K362Q/E367N/R373K





1. Relative activity was calculated as activity of the variant/activity of Rd3BB (SEQ ID NO: 13) (encoded by SEQ ID NO: 11).


2. Variant # 625 (Rd7BB) has the polynucleotide sequence of SEQ ID NO: 43 and polypeptide sequence of SEQ ID NO: 44.


3. − = <1.5 relative activity to Rd3BB (SEQ ID NO: 13); + = 1.5 to 5 relative activity over Rd3BB (SEQ ID NO: 13); ++ = >5 to 10 relative activity over Rd3BB (SEQ ID NO: 13); and +++ = >10 relative activity over Rd3BB (SEQ ID NO: 13).













TABLE 2.9







Relative Activity of GLA Variants After No Challenge (NC) or Challenge at the Indicated pH or Condition












Variant

pH
pH
Amino acid differences relative to SEQ ID NO: 5
SEQ


#
NC
3.5
7.5
(WT GLA)
ID NO:















638



T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/H271A/
678






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


639



M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247Y/H271A/Q302K/
679






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


640
+
+
+
T10P/M39E/E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/
680






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


641



T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/S266P/
681






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


642
+
+
+
T10P/E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/
682






H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/W368A/






R373K/M392T


643
+

+
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
683






L316D/M322I/R325S/A337P/K362Q/E367N/R373K/M392T


644



L44R/S47T/Y92H/S166P/K206A/F217R/L237P/N247D/H271A/Q302K/
684






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


645
+
+

L44R/S47T/Y92H/S166P/P174S/K206A/F217R/N247D/H271A/Q302K/
685






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


646



L44R/S47T/Y92H/G113C/S166P/K206A/F217R/N247D/H271A/Q302K/
686






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


647



L14F/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
687






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


648
+
+
+
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/
46






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


649
+
+
+
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/
689






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


650
+
+
+
R7H/T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
690






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


651
+
+
+
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
691






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


652
+
+

L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/A261G/H271A/
692






Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


653
+
+
+
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/H271A/
693






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


654
+
+
+
R7S/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
694






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


655
+


L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/A261G/H271A/
695






Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


656
+
+
+
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/A261G/
696






H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


657
+
+
+
L44R/S47T/P67T/Y92H/S166P/K182N/K206A/F217R/N247D/H271A/
697






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


658
+
+
+
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/H271A/
698






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


659



L44R/S47T/W64L/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
699






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


660
+
+

M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/H271A/
700






Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


661



L44R/S47T/Y92H/S166P/W195C/K206A/F217R/N247D/H271A/Q302K/
701






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


662
+
+
+
L44R/S47T/Y92H/S166P/K206A/F217R/V2381/N247D/H271A/Q302K/
702






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


663
+
+

E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/H271A/
703






Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


664



T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/Q252H/M253R/
704






A254E/A261G/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/






M392T


665
+
+
+
R7C/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
705






L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


666
+
+

L44R/S47T/Y92H/S166P/K206A/F217R/P228L/N247D/H271A/Q302K/
706






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


667
+
+
+
D30G/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
707






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


668
+
+
+
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
708






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


669
+


L44R/S47T/Y92H/S166P/K206A/F217R/N247D/P262S/H271A/Q302K/
709






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


670
+
+
+
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/N305L/
710






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


671
++
++
+
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
711






L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


672
+
+

L44R/S47T/Y92H/D144Y/S166P/K206A/F217R/N247D/H271A/Q302K/
712






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


673
+
+

L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/L316D/
713






M322I/A337P/K362Q/E367N/R373K/N377Y/M392T


674



L44R/S47T/Y92H/S166P/K206A/F217R/P234H/N247D/H271A/Q302K/
714






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


675
+
+

L44R/S47T/M65V/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
715






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


676
+
+
+
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
716






L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


677
+
+

L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M253W/H271A/S273D/
717






P274S/K277R/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


678



L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M253W/A257G/H271A/
718






K277R/Q281L/Q302K/L316D/A319D/M322I/A337P/K362Q/E367N/R373K/






M392T


679
+
+
+
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
719






Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


680
+
+
+
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
720






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


681



R7P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
721






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


682
+
+
+
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/L316Y/
722






M322I/A337P/K362Q/E367N/R373K/M392T


683
+
+

M39E/E43D/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/
723






M253W/H271A/S273D/Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/






R373K/M392T


684
+
+
+
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/N305L/
724






L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


685
+


E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M253W/A257G/
725






H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/W368A/






R373K/M392T


686


+
T10P/E17G/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
726






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


687
+


L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q290R/Q302K/
727






L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


688
+
+

L44R/S47T/Y92H/S166P/K206A/F217R/P228Q/N247D/H271A/Q302K/
728






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


689
+
+
+
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/N305L/
729






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


690
+


T10P/L44R/S47T/Y92H/M156V/S166P/K206A/F217R/N247D/H271A/
730






Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


691
+
+
+
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
731






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


692



L44R/S47T/Y92H/S166P/K206A/F217R/N247D/W256L/H271A/Q302K/
732






L316D/M322I/A337P/K362Q/E367N/R373K/M392T


693
+
+
+
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/L316D/
733






M322I/A337P/K362Q/E367N/W368A/R373K/M392T





1. Relative activity was calculated as activity of the variant/activity of Rd7BB (SEQ ID NO: 44) (encoded by SEQ ID NO: 43).


2. − = <0.5 relative activity to Rd7BB (SEQ ID NO: 44); + = >0.5 to 1.5 relative activity over Rd7BB (SEQ ID NO: 44); and ++ = >1.5 relative activity over Rd7BB (SEQ ID NO: 44);






Example 3
In Vitro Characterization of GLA Variants
Production of GLA in Yeast

In order to produce GLA-containing supernatant, replica HTP-cultures of GLA were grown as described in Example 2. Supernatants from replica cultures (n=12-36) were combined prior to further analysis.


Production of GLA in HEK293T Cells

Secreted expression of GLA variants in mammalian cells was performed by transient transfection of HEK293 cells. Cells were transfected with GLA variants (SEQ ID NOS:3, 4, 9, 12, 17, 20, 23, and 41) fused to an N-terminal synthetic mammalian signal peptide and subcloned into the mammalian expression vector pLEV113 as described in Example 1. HEK293 cells were transfected with plasmid DNA and grown in suspension for 4 days using techniques known to those skilled in the art. Supernatants were collected and stored at 4° C.


Example 4
Purification of GLA Variants

Purification of GLA Variants from Mammalian Cell Supernatants


GLA variants were purified from mammalian culture supernatant essentially as known in the art (See, Yasuda et al., Prot. Exp. Pur. 37, 499-506 [2004]). Concanavalin A resin (Sigma Aldrich) was equilibrated with 0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgC2, CaC2, and MnCl2 pH 6.0 (Con A binding buffer). Supernatant was diluted 1:1 with binding buffer and loaded onto the column. The column was washed with 15 volumes of Concanavalin A binding buffer, and samples were eluted by the addition of Concanavalin A binding buffer including 0.9 M methyl-α-D-mannopyranoside and 0.9 M methyl-α-D-glucopyranoside. Eluted protein was concentrated and buffer exchanged three times using a Centricon® Plus-20 filtration unit with a 10 kDa molecular weight cut off (Millipore) into ThioGal binding buffer (25 mM citrate-phosphate, 0.1 M NaCl, pH 4.8). Buffer exchanged samples were loaded onto a Immobilized-D-galactose resin (Pierce) equilibrated with ThioGal binding buffer. The resin was washed with six volumes of ThioGal binding buffer and eluted with 25 mM citrate phosphate, 0.1 M NaCl, 0.1 M D-galactose, pH 5.5. Eluted samples were concentrated using a Centricon® Plus-20 filtration unit with a 10 kDa molecular weight cut off. Purification resulted in between 2.4-10 g of purified protein/ml of culture supernatant based on Bradford quantitation.


SDS-PAGE Analysis of GLA Variants

Samples of yeast culture supernatant and mammalian cell culture supernatant and purified GLA were analyzed by SDS-PAGE. In the yeast supernatants, GLA levels were too low to be detected via this method. Bands corresponding to the ˜49 kDa predicted GLA molecular weight were found in both mammalian cell culture supernatants and purified GLA samples.


Immunoblot Analysis of GLA Variants

Samples of yeast supernatant and mammalian cell culture supernatant were analyzed by immunoblot. Briefly, samples were separated via SDS-PAGE. Protein was transferred to a PVDF membrane using an iBlot dry blot system (Life Technologies). The membrane was blocked with Odyssey blocking buffer (TBS) (LI-COR) for 1 h at RT and probed with a 1:250 dilution of rabbit α-GLA IgG (Thermo-Fischer) in Odyssey blocking buffer with 0.2% Tween® 20 for 14 h at 4° C. The membrane was washed 4×5 min with Tris-buffered saline+0.1% Tween® 20 and probed with a 1:5000 dilution of IRDye800CW donkey α-rabbit IgG (LI-COR) in Odyssey blocking buffer with 0.2% Tween® 20 and 0.01% SDS for 1 hr at RT. The membrane was washed 4×5 min with Tris-buffered saline+0.1% Tween® 20, and analyzed using an Odyssey Imager (LI-COR). Bands corresponding to the ˜49 kDa predicted GLA molecular weight were found in both the mammalian cell culture and yeast supernatants. In S. cerevisiae expressed samples, mutants containing the mutation E367N ran at a slightly higher MW. This mutation introduces a canonical NXT N-linked glycosylation site (where X is any amino acid except P) and the possible introduction of an additional N-linked glycan may account for the higher MW.


Example 5
In Vitro Characterization of GLA Variants

Optimization of Signal Peptide for Secreted Expression of GLA by S. cerevisiae



S. cerevisiae transformed with Mfleader-GLA (SEQ ID NO:7), SP-GLA (SEQ ID NO:36) or a vector control were grown in HTP as described in Example 2. Cultures were grown for 48-120 h prior to harvest of the supernatant and analysis (n=6) as described in Example 2. FIG. 1 provides a graph showing the relative activity of different GLA constructs in S. cerevisiae after 2-5 days of culturing. As indicated in this Figure, SP-GLA (SEQ ID NO:36) produced a high level of active enzyme that saturated after three days of growth.


pH Stability of GLA Variants Expressed in S. cerevisiae


GLA variants were challenged with different buffers to assess the overall stability of the enzyme. First, 50 μL of supernatant from a GLA variant yeast culture and 50 uL of McIlvaine buffer (pH 2.86-9.27) or 200 mM sodium carbonate (pH 9.69) were added to the wells of a 96-well round bottom plate (Costar #3798, Corning). The plates were sealed and incubated at 37° C. for 1 h. For the assay, 50 μL of challenged supernatant was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37° C. for 60-180 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG. 2 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants after incubation at various pHs.


Thermostability of GLA Variants Expressed in S. cerevisiae


GLA variants were challenged at various temperatures in the presence and absence of 1 M 1-deoxygalactonojirimycin (Migalastat; Toronto Research Chemicals) to assess the overall stability of the enzyme. First, 50 μL of supernatant from a GLA variant yeast culture and 50 uL of McIlvaine buffer (pH 7.65)+/−2 mM 1-deoxygalactonojirimycin were added to the wells of a 96-well PCR plate (Biorad, HSP-9601). The plates were sealed and incubated at 30-54° C. for 1 h using the gradient program of a thermocycler. For the assay, 50 μL of challenged supernatant was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37° C. for 90 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG. 3 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants after incubation at various temperatures.


Serum Stability of GLA Variants Expressed in S. cerevisiae


To assess the relative stability of variants in the presence of blood, samples were exposed to serum. First, 20 μL of supernatant from a GLA variant yeast culture and 0-80 μL of water and 0-80 L of bovine serum were added to the wells of a 96-well round bottom plate (Costar #3798, Corning). The plates were sealed and incubated at 37° C. for 1 h. For the assay, 50 μL of challenged supernatant was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37° C. for 90 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG. 4 provides graphs showing the absolute (Panels A and B) and relative (Panels C and D) activity of GLA variants after challenge with various percentages of serum.


Relative Activities of GLA Variants Expressed in HEK293T Cells

Supernatants from GLA variants expressed in HEKT293T cells were serially diluted 2× with supernatant from a non GLA expressing yeast culture. Dilutions (50 μL) were mixed with 25 μL of 4 mM MUGal in McIlvaine Buffer pH 4.8 and 25 μL of 1 M citrate buffer pH 4.3 in a Corning® 96-well, black, opaque bottom plate. The reactions were mixed briefly and incubated at 37° C. for 60 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG. 5 provides a graph showing the relative activity of GLA variants expressed in HEK293T cells. Supernatants from cells transfected with variant GLA enzymes showed markedly higher hydrolase activities compared to the WT enzymes, and much more activity per volume than was seen in S. cerevisiae expression.


pH Stability of GLA Variants Expressed in HEK293T Cells

GLA variants were challenged with different buffers to assess their overall stability. Supernatants from mammalian cell cultures were normalized to equal activities by dilution with supernatant from a non GLA expressing culture. Normalized supernatants (50 μL) and 50 uL of McIlvaine buffer (pH 4.06-8.14) were added to the wells of a 96-well round bottom plate (Costar #3798, Corning). The plates were sealed and incubated at 37° C. for 3 h. For the assay, 50 μL of challenged supernatant was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37° C. for 3 h, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG. 6 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various pHs.


All enzymes were found to be more stable versus pH challenges when compared to WT GLA expressed in S. cerevisiae (compare with FIG. 2). This difference is possibly due to differential glycosylation between expression hosts. However, it is not intended that the present invention be limited to any particular mechanism or theory. Mutant enzymes had broader pH stability profiles compared to the WT enzyme expressed in HEK293T.


Thermostability of GLA Variants Expressed in HEK293T Cells

GLA variants were challenged at various temperatures in the presence and absence of 1 M 1-deoxygalactonojirimycin (Migalastat) to assess their overall stability. Supernatants from mammalian cell cultures were normalized to approximately equal activities by dilution with supernatant from a non GLA expressing culture. Diluted supernatants were added to the wells of a 96-well PCR plate (Biorad, HSP-9601). The plates were sealed and incubated at 30-54° C. for 1 h using the gradient program of a thermocycler. For the assay, 20 μL of challenged supernatant was mixed with 30 μL of 1 M citrate buffer pH 4.3 and 50 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37° C. for 90 minutes, prior to quenching with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm). FIG. 7 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various temperatures. As shown in this Figure, all of the enzymes were more stable after temperature challenges when compared to WT GLA expressed in S. cerevisiae (compare with FIG. 2), likely due to differential glycosylation between expression hosts. In the GLA variants (SEQ ID NOS:10 and 13) the Tm of the enzyme was increased by 2 and 4° C. respectively. Addition of Migalastat increased the Tm by 5.5° C., however at a 0.2 M final concentration in the assay, activity in the Migalastat treated sample was reduced by ˜60%.


Activity of WT GLA and GLA Variants on an Alternative Substrate

To confirm that improved activity in MUGal hydrolysis corresponded to more native substrates, mammalian cell-expressed GLA variants were assayed using N-Dodecanoyl-NBD-ceramide trihexoside (NBD-GB3) as substrate. HEK293T culture supernatant (10 μL), 100 mM sodium citrate pH 4.8 (80 μL), and NBD-GB3 (0.1 mg/ml) in 10% ethanol (10 μL) were added to microcentrifuge tubes. Samples were inverted to mix, and incubated at 37° C. for 1 h. The reaction was quenched via addition of 50 μL methanol, diluted with 100 μL chloroform, vortexed and the organic layer was isolated for analysis. The organic phase (10 μL) was spotted onto a silica plate and analyzed by thin layer chromatography (chloroform:methanol:water, 100:42:6), detecting the starting material and product using a 365 nm UV lamp. Significant conversion was observed only with SEQ ID NO:13, confirming that the variant exhibits improved activity, as compared to the WT GLA.


Specific Activity of GLA Variants

GLA variants purified as described in Example 4, were evaluated for their specific activity. Between 0-0.25 ng of purified enzyme was added to 4 mM MUGal in McIlvaine buffer pH 4.8 (final pH of 4.8). Samples were incubated for 60 min at 37° C. and quenched via addition of 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (Ex. 355 nm, Em. 448 nm), and correlated to absolute amounts of 4-methylumbelliferone through the use of a standard curve.


pH Stability of Purified GLA Variants Over Time

To confirm that purified GLA variants show the desired pH stability observed after expression in yeast, WT GLA (SEQ ID NO:5) and SEQ ID NO:42 were incubated in acidic or basic buffers and analyzed for residual activity. GLA variants (200 ng) were added to McIlvaine buffer pH 4.1 or 7.5 and incubated for 0-24 h at 37° C. Samples (50 μL) were added to a mixture of 25 uL 1M citric acid pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8, and incubated at 37° C. for 1 h. Samples were quenched with 100 μL of 1 M sodium carbonate, diluted 1:4 in 1 M sodium carbonate and analyzed by fluorescence spectroscopy (Ex. 355, Em. 448). SEQ ID NO:42 was considerably more stable under both acidic and basic challenge conditions confirming that stability advances developed in yeast translated to the protein expressed in mammalian cells (See FIG. 8 for graphs of the results).


Thermostability of Purified GLA Variants Expressed in HEK293T Cells

The thermostability of WT GLA (SEQ ID NO:5) and SEQ ID NO:42 were determined to assess their overall stability. Purified enzyme as described in Example 4 was diluted to 20 μg/ml in 1×PBS with 1× Sypro Orange (Thermo Fischer Scientific), and added to a 96-well PCR plate (Biorad, HSP-9601). The plates were heated from 30 to 75° C. at 0.5° C./min on a RT-PCR machine and Sypro Orange fluorescence was monitored. Under these conditions WT GLA melted at 37° C., while SEQ ID NO:42 melted at 55° C.


Example 6
In Vivo Characterization of GLA Variants
Serum Pharmacokinetics of Purified GLA Variants

Purified GLA variants produced as described in Example 4 were assessed for stability in the serum of live rats. WT GLA (SEQ ID NO:5) or SEQ ID NO:42 at 1 mg/ml were administered intravenously at 1 ml/kg to three naïve jugular vein cannulated Sprague-Dawley rats (7-8 weeks old) each. Prior to administration and at 5, 15, 30, 60, 120, and 240 minutes post-administration, 200 μL of blood was collected from each rat in an EDTA tube and centrifuged at 4° C. and 6000 rpm to generate >80 μL of serum per sample. Samples were frozen and stored on dry ice prior to analysis. For analysis, serum (10 μL) was added to 40 μL of 5 mM MUGal in McIlvaine buffer pH 4.4, and incubated at 37° C. for 1 h. Samples were quenched with 50 μL of 1 M sodium carbonate, diluted 1:100 in 1 M sodium carbonate and analyzed by fluorescence spectroscopy (Ex. 355, Em. 448). Four hours post-administration SEQ ID NO:42 retained 15.3% of maximal activity, while WT GLA retained only 0.66% (See, FIG. 9).


Example 7
Deimmunization of GLA

In this Example, experiments conducted to identify diversity that would remove predicted T-cell epitopes from GLA are described.


Identification of Deimmunizing Diversity:

To identify mutational diversity that would remove T-cell epitopes, computational methods were used to identify GLA subsequences that were predicted to bind efficiently to representative HLA receptors. In addition, experimental searches for amino acid mutations were conducted, particularly for mutations that do not affect GLA activity (e.g., in the assays described in Example 2). The amino acid sequences of active variants were then analyzed for predicted immunogenicity using computational methods.


Computational Identification of Putative T-Cell Epitopes in a WT GLA:

Putative T-cell epitopes in a WT GLA (SEQ ID NO:5) were identified using the Immune Epitope Database (IEDB; Immune Epitope Database and Analysis Resource website) tools, as known in the art and proprietary statistical analysis tools (See e.g., iedb.org and Vita et al., Nucl. Acids Res., 38(Database issue):D854-62 [2010]. Epub 2009 Nov. 11]). The WT GLA was parsed into all possible 15-mer analysis frames, with each frame overlapping the last by 14 amino acids. The 15-mer analysis frames were evaluated for immunogenic potential by scoring their 9-mer core regions for predicted binding to eight common class II HLA-DR alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501) that collectively cover nearly 95% of the human population (See e.g., Southwood et al., J. Immunol., 160:3363-3373 [1998]), using methods recommended on the IEDB website. Potential T-cell epitope clusters contained within the enzyme (i.e., sub-regions contained within GLA which have an unusually high potential for immunogenicity) were identified using statistical analysis tools, as known in the art. The identified T-cell epitope clusters were screened against the IEDB database of known epitopes. These screens identified five putative T-cell epitopes in the WT enzyme. These epitopes are referred to as TCE-I, II, III, IV, and V below.


Design of Deimmunizing Libraries:

First, the sequences of active GLA mutants identified in Example 2 are assessed for the presence of T-cell epitopes. Mutations identified to potentially reduce binding to the HLA-DR alleles are incorporated into a recombination library. Additional libraries are prepared using saturation mutagenesis of every single amino acid within the five T-cell epitopes. Hits from these libraries are subjected to further rounds of saturation mutagenesis, HTP screening, and recombination to remove all possible T-cell epitopes.


Construction and Screening of Deimmunizing Libraries:

Combinatorial and saturation mutagenesis libraries designed as described above were constructed by methods known in the art, and tested for activity in an unchallenged assay as described in Example 2. Active variants were identified and sequenced. Their activities and mutations with respect to WT GLA are provided in the table below.


Identification of Deimmunizing Diversity:

Active variants were analyzed for their levels of predicted immunogenicity by evaluating their binding to the eight common Class II HLA-DR alleles as described above. The total immunogenicity score and immunogenic hit count are shown in Table 7.1. The total immunogenicity score (TIS) reflects the overall predicted immunogenicity of the variant (i.e., a higher score indicates a higher level of predicted immunogenicity). The immunogenic “hit count” (IHC) indicates the number of 15-mer analysis frames with an unusually high potential for immunogenicity (i.e., a higher score indicates a higher potential for immunogenicity). Mutations resulting in a lower total immunogenicity score and/or an immunogenic hit count less than that of the reference sequence were considered to be potential “deimmunizing mutations”. A collection of the most deimmunizing mutations were recombined to generate a number of variants that were active and predicted to be significantly less immunogenic than WT GLA. In the following Table, total immunogenicity score (TIS) and immunogenic hit count (IHC) are provided.









TABLE 7.1







Total Immunogenicity Score (TIS), and Immunogenic Hit Count (IHC) for GLA Variants











Variant
SEQ ID





#
NO:
Active Mutations
TIS
IHC















5
WT GLA
450
38


33
79
A199H/E367S
468
47


34
80
A337P
444
38


1
47
A337S
449
38


35
81
A339S
450
38


36
82
A350G
450
38


296
337
A66T/K206A/F217R/L316D/M322I/A337P/K343G/A350G/E367N/R373K
429
38


200
244
C143A/K206A
429
38


201
245
C143T/K206A
429
38


202
246
C59A/K206A
427
38


37
83
D105A
458
38


38
84
D105S
462
38


39
85
D124N/E147G/N161K/R162Q/T163V/R165A/I167S/V168I/Y169V/
425
35




S170—/M177S/F217E


516
557
D2E/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




Q326G/A337P/K362Q/E367N/R373K


517
558
D2Q/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


667
707
D30G/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
345
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


40
86
D396R
451
38


41
87
D396T
452
38


42
88
E367N
462
43


43
89
E367T
462
45


44
90
E387K
460
38


45
91
E387Q
457
38


46
92
E387R
457
38


47
93
E387T
459
38


48
94
E40D
445
33


518
560
E40D/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
390
24




A337P/K362Q/E367N/R373K


519
561
E40S/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
407
25




A337P/K362Q/E367N/R373K


2
48
E43D
450
37


3
49
E43D/E48D
449
37


4
50
E43D/E48D/I208V/N247D/Q299R/Q302K/R373K/I376V
434
36


5
51
E43D/E48D/I208V/R373K
429
36


6
52
E43D/E48D/I208V/R373K/I376V
428
36


7
53
E43D/E48D/N247D/Q299R/Q302K/R373K/I376V
448
36


8
54
E43D/E48D/N247D/Q302K/R373K
442
36


9
55
E43D/E48D/Q302K/R373K/I376V
442
36


10
56
E43D/I208V/N247D
435
37


11
57
E43D/I208V/N247D/Q299R/R373K/I376V
435
36


12
58
E43D/I208V/Q299R/R373K/I376V
436
36


663
703
E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/
315
1




H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/




R373K/M392T


685
725
E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M253W/
334
7




A257G/H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/




E367N/W368A/R373K/M392T


634
674
E43D/L44R/Y92E/S166P/K206A/F217R/N247D/Q302K/L316D/
362
21




M322I/A337P/K362Q/E367N/R373K


635
375
E43D/L44R/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/
378
21




M322I/A337P/K362Q/E367N/R373K


636
376
E43D/L44R/Y92N/S166P/K206A/F217R/N247D/Q302K/L316D/
366
21




M322I/A337P/K362Q/E367N/R373K


633
673
E43D/L44R/Y92S/S166P/K206A/F217R/N247D/Q302K/L316D/
365
21




M322I/A337P/K362Q/E367N/R373K


13
59
E43D/N247D/R373K/I376V
442
36


14
60
E43D/R373K/I376V
443
36


637
377
E43Q/L44R/Y92E/S166P/K206A/F217R/N247D/Q302K/L316D/
370
21




M322I/A337P/K362Q/E367N/R373K


15
61
E48D/I208V/Q299R/Q302K/R373K
437
37


16
62
E48D/R373K/I376V
443
37


17
63
E48G/R373K
444
37


49
95
F180R
454
38


50
96
F180S
449
38


51
97
F198S
450
38


52
98
F217D
450
38


53
99
F217R
450
38


18
64
F217S
452
38


54
100
F352I
450
38


55
101
F352V/F365I
447
38


56
102
F365I
447
38


57
103
F365K
446
38


58
104
F365E
448
38


59
105
F365R
447
38


60
106
F365T
436
38


61
107
F365V
447
38


62
108
G303Q/R373V
465
38


63
109
H155A
451
38


64
110
H155E
455
41


65
111
H155R
452
39


66
112
H155T
449
38


213
255
H15Q/K206A
429
38


67
113
H375E
437
36


68
114
H84S
450
38


69
115
I102L
450
38


70
116
I102L/L394V
449
37


71
117
I123T/T369N
449
38


72
118
I167V
438
37


19
65
I208V/N247D/Q299R/Q302K/R373K/I376V
435
37


20
66
I208V/N247D/Q299R/R373K/I376V
435
37


21
67
I208V/N247D/R373K/I376V
428
37


22
68
I208V/Q299R/I376V
436
37


23
69
I208V/Q302K/R373K/I376V
429
37


24
70
I376V
443
37


73
10
K206A
429
38


196
240
K206A/A350G
429
38


197
241
K206A/A350G/K362Q/T369A
413
38


198
242
K206A/A350G/T369D
426
38


199
243
K206A/A350G/T369S
429
38


203
247
K206A/E367A/T369D
439
42


204
248
K206A/E367D
427
38


205
21
K206A/E367D/T369D
419
37


206
18
K206A/E367N
441
43


207
249
K206A/E367N/R373K
430
38


208
250
K206A/E367N/R373K/I376V
429
38


209
251
K206A/E367P/T369D
430
38


297
338
K206A/F217R/G230V/N247D/Q302K/M322I/E367N/T369S/R373K
453
42


233
274
K206A/F217R/N247D/L316D/A350G/E367D/T369D
416
37


298
339
K206A/F217R/N247D/L316D/M322I/A337P/A350G/K362Q/E367N/
420
37




R373K


299
340
K206A/F217R/N247D/Q249H/Q302K/M322I/K343G/A350G/E367T/
434
40




R373K/L397F


234
275
K206A/F217R/N247D/Q302K/A350G/E367D/T369D
418
37


235
276
K206A/F217R/N247D/Q302K/L316D/A337P/A350G/E367D/T369D
410
37


236
277
K206A/F217R/Q302K/E367D/T369D
419
37


237
278
K206A/F217R/Q302K/L316D/A337P/A350G/E367D/T369D
411
37


210
252
K206A/F365L/E367N
439
41


211
253
K206A/F365L/E367N/I376V
435
40


212
254
K206A/F365L/E367N/R373K/I376V
435
40


238
279
K206A/I208V/M322V/K343G/F365L/R373K/I376V
415
37


239
280
K206A/I208V/R221K/N247D/M322I/K343D/F365L/R373K/I376V
425
37


300
341
K206A/I208V/R221T/N247D/M322V/K343G/E367N/R373K
424
38


214
256
K206A/K343D/F365L/E367N
433
41


215
257
K206A/K343G
424
38


216
258
K206A/K343G/F365L/E367N/R373K
431
40


240
281
K206A/L269I/P349L/R373K
428
42


217
289
K206A/L316D
427
38


218
13
K206A/M322I/E367N/R373K
442
38


301
342
K206A/M322I/E367N/R373K
442
38


219
260
K206A/M322I/R373K
435
37


302
343
K206A/M322V/K343G/E367N/R373K
425
38


220
261
K206A/M322V/R373K/I376V
422
37


221
262
K206A/M390I
414
33


303
344
K206A/N247D/M322I/A337E/K343D/F365L/E367N/R373K/I376V
440
40


241
282
K206A/N247D/M322V/K343D/R373K/I376V
415
37


242
283
K206A/N247D/M322V/K343G/F365L/R373K
415
37


243
284
K206A/N247D/Q302K/A337P/K343G/A350G
417
38


244
285
K206A/N247D/Q302K/L316D/A350G
426
38


245
286
K206A/N247D/Q302K/M322V/F365L/R373K/I376V
419
37


222
263
K206A/P228Q/T369D
426
38


223
264
K206A/Q302K/A337P/A350G/K362Q
408
38


246
287
K206A/Q302K/L316D/A337P
421
38


304
345
K206A/Q302K/L316D/M322I/A337P/A350G/K362Q/E367N/T369S/
431
42




R373K


305
346
K206A/Q302K/L316D/M322I/A337P/K343D/E367N/T369S/R373K
438
42


224
265
K206A/Q302K/M322V/E367N
441
43


247
288
K206A/R221K/N247D/M322V/K343D/R373K
416
37


306
347
K206A/R221K/N247D/Q302K/M322I/E367N/R373K
441
38


307
348
K206A/R221K/Q302K/M322I/K343G/E367N/R373K/I376V
436
38


226
267
K206A/R221T/F365L
427
38


248
289
K206A/R221T/M322V/K343G/R373K
418
37


249
290
K206A/R221T/M322V/R373K
423
37


227
268
K206A/R325H
429
38


228
269
K206A/R373K
423
37


229
270
K206A/R373K/I376V
422
37


230
271
K206A/S374R
433
40


231
272
K206A/T369D
426
38


232
273
K206A/T369S
429
38


192
236
K206E
429
38


193
237
K206G
429
38


74
119
K206M
458
44


75
120
K206Q
450
38


76
24
K206R
450
38


194
238
K206R
450
38


195
239
K206S
429
38


77
121
K206T/V359S
437
44


78
122
K343D
444
38


79
123
K343G
445
38


80
124
K362Q
435
38


81
125
K362R
449
38


82
126
K36D
452
38


83
127
K36E
450
38


25
71
K36Q
450
38


84
128
K395*
432
34


85
129
K395G
448
37


86
130
K395P
448
37


87
131
K395R
451
38


88
132
K395S
450
38


89
133
K395T
448
37


90
134
K96I
433
36


250
291
K96I/K206A/F217R
412
36


308
349
K96I/K206A/F217R/M322I/E367N/T369S/R373K
434
40


251
292
K96I/K206A/F217R/N247D
411
36


252
293
K96I/K206A/F217R/N247D/A350G/E367D/T369D
401
35


253
294
K96I/K206A/F217R/N247D/Q302K/E316D/A337P/E367D/T369D
393
35


309
350
K96I/K206A/F217R/N247D/Q302K/M322I/A337P/K343G/A350G/
413
36




E367N/R373K


310
351
K96I/K206A/N247D/M322I/A350G/E367N/T369S/R373K
433
40


311
352
K96I/K206A/N247D/Q302K/E316D/M322I/A337P/A350G/E367N/
425
40




T369S/R373K


312
353
K96I/K206A/N247D/Q302K/E316D/M322I/A337P/A350G/K362Q/
413
40




E367N/T369S/R373K


91
135
K96E
434
36


92
136
K96R
443
37


93
137
K96R/E397V
442
36


94
138
E100F
442
38


313
354
L100F/A125S/K206A/I208V/R221K/Q302K/M322I/K343G/E367N/
429
38




R373K


254
295
L100F/K206A
421
38


314
355
L100F/K206A/I208V/N247D/Q302K/M322V/K343D/E367N/R373K/
414
38




I376V


315
356
L100F/K206A/I208V/Q302K/M322V/F365L/E367N/R373K/I376V
427
40


316
357
L100F/K206A/I208V/R221K/M322V/K343D/E367N/R373K
416
38


317
358
L100F/K206A/I208V/R221K/M322V/K343D/F365L/E367N/R373K
422
40


255
296
L100F/K206A/I208V/R221K/N247D/Q302K/M322I/K343D/F365L/
417
37




I376V


256
297
L100F/K206A/I208V/R221K/N247D/Q302K/M322V/K343D/F365L/
405
37




I376V


318
359
L100F/K206A/I208V/R221T/M322V/E367N/R373K/I376V
421
38


257
298
L100F/K206A/I208V/R221T/N247D/K343D/F365L/I376V
405
37


258
299
L100F/K206A/I208V/R221T/Q302K/M322I/K343D/I376V
420
37


319
360
L100F/K206A/M322I/E367N/R373K/I376V
433
38


259
300
L100F/K206A/M322V/F365L/R373K/I376V
412
37


260
301
L100F/K206A/N247D/F365L/R373K/I376V
411
37


261
302
L100F/K206A/N247D/M322V/K343D/I376V
407
37


320
361
L100F/K206A/N247D/Q302K/M322I/E367N/R373K
433
38


321
362
L100F/K206A/R221K/N247D/M322I/K343G/E367N/R373K
428
38


262
303
L100F/K206A/R221K/N247D/Q302K/M322V/F365L/R373K/I376V
411
37


263
304
L100F/K206A/R221K/N247D/Q302K/M322V/I376V
413
37


264
305
L100F/K206A/R221K/N247D/Q302K/M322V/K343D/R373K/I376V
407
37


265
306
L100F/K206A/R221K/R373K/I376V
414
37


266
307
L100F/K206A/R221T/M322I/K343E/F365L/R373K
419
37


267
308
L100F/K206A/R221T/N247D/Q302K/K343D/F365L/R373K
406
37


322
363
L100F/K206A/R221T/Q302K/M322I/K343D/E367N/R373K
428
38


268
309
L100F/K206A/R373K/I376V
414
37


323
364
L100F/L160I/K206A/R221K/M322V/E367N/R373K
424
42


647
387
L14F/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
345
8




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


95
139
L158A
437
35


96
140
L158I
458
42


97
141
L158M
450
40


98
142
L158R
431
35


99
143
L23M
450
38


324
365
L23S/K206A/M322I/E367N/R373K
442
38


100
144
L23T
450
38


101
145
L316D
448
38


102
146
L316E
448
38


269
310
L37I/K206A/R221K/N247D/M322I/R373K
434
37


103
147
L384F
448
35


104
148
L386V
436
31


105
149
L394A
449
37


106
150
L394R
450
38


107
151
L394S
450
38


108
152
L394T
449
37


109
153
L397*
442
36


110
154
L397D
449
37


111
155
L397H
450
38


112
156
L397I
449
37


113
157
L397Q
449
37


114
158
L397R
449
37


115
159
L397T
449
37


116
160
L398E
449
37


117
161
L398G
450
38


118
162
L398N
449
37


119
163
L398Q
450
38


120
164
L398R
449
37


368
409
L44A/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K


362
403
L44C/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K


360
401
L44E/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K


374
415
L44M/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
33




E367N/R373K


370
411
L44Q/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K


398
439
L44R/A159S/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


520
561
L44R/A77S/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


270
311
L44R/C143Y/K206A/A337P/A350G
430
38


521
562
L44R/D52N/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


271
312
L44R/E187G/K206A/A337P/A350G
430
38


522
563
L44R/E56K/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


382
423
L44R/H94N/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
30




K362Q/E367N/R373K


386
427
L44R/H94R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


272
313
L44R/K206A
436
38


273
314
L44R/K206A/E367D/T369D
426
37


274
315
L44R/K206A/F217R/A350G
436
38


275
316
L44R/K206A/F217R/N247D/A337P
429
38


439
480
L44R/K206A/F217R/N247D/H271A/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


425
466
L44R/K206A/F217R/N247D/H271E/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


436
477
L44R/K206A/F217R/N247D/H271G/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


433
474
L44R/K206A/F217R/N247D/H271Q/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


452
493
L44R/K206A/F217R/N247D/H271R/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


430
471
L44R/K206A/F217R/N247D/H271T/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


441
482
L44R/K206A/F217R/N247D/H271V/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


435
476
L44R/K206A/F217R/N247D/I258L/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


450
491
L44R/K206A/F217R/N247D/I258M/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


442
483
L44R/K206A/F217R/N247D/L255A/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


429
470
L44R/K206A/F217R/N247D/L255C/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


424
465
L44R/K206A/F217R/N247D/L255E/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


443
484
L44R/K206A/F217R/N247D/L255S/Q302K/L316D/M322I/A337P/
N.D.
35




K362Q/E367N/R373K


449
490
L44R/K206A/F217R/N247D/L255T/Q302K/L316D/M322I/A337P/
N.D.
35




K362Q/E367N/R373K


432
473
L44R/K206A/F217R/N247D/L255V/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


440
481
L44R/K206A/F217R/N247D/L263C/Q302K/L316D/M322I/A337P/
N.D.
33




K362Q/E367N/R373K


437
478
L44R/K206A/F217R/N247D/L263E/Q302K/L316D/M322I/A337P/
N.D.
29




K362Q/E367N/R373K


445
486
L44R/K206A/F217R/N247D/L263F/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


427
468
L44R/K206A/F217R/N247D/L263G/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


447
488
L44R/K206A/F217R/N247D/L263W/Q302K/L316D/M322I/A337P/
N.D.
33




K362Q/E367N/R373K


276
317
L44R/K206A/F217R/N247D/L316D/A350G/E367D/T369D
417
37


277
318
L44R/K206A/F217R/N247D/L316D/A337P/E367D/T369D
417
37


278
319
L44R/K206A/F217R/N247D/L316D/A350G/E367D/T369D
423
37


325
366
L44R/K206A/F217R/N247D/L316D/M322I/A337P/K343G/K362Q/
422
37




E367N/R373K


446
487
L44R/K206A/F217R/N247D/M259A/Q302K/L316D/M322I/A337P/
N.D.
31




K362Q/E367N/R373K


426
467
L44R/K206A/F217R/N247D/M259E/Q302K/L316D/M322I/A337P/
N.D.
30




K362Q/E367N/R373K


428
469
L44R/K206A/F217R/N247D/M259S/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


451
492
L44R/K206A/F217R/N247D/M259V/Q302K/L316D/M322I/A337P/
N.D.
35




K362Q/E367N/R373K


444
485
L44R/K206A/F217R/N247D/M259W/Q302K/L316D/M322I/A337P/
N.D.
30




K362Q/E367N/R373K


279
320
L44R/K206A/F217R/N247D/Q302K/A350G
435
38


326
367
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/K362Q/E367N/
427
37




R373K


513
554
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K/D396*


512
553
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K/K395*


508
549
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/L384A


477
518
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
34




E367N/R373K/L384W


474
515
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
35




E367N/R373K/L386F


502
543
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/L386S


460
501
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/L386T


515
556
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K/L394*


511
552
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K/L397*


500
541
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K/M390A


486
527
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
35




E367N/R373K/M390C


490
531
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390D


476
517
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390E


494
535
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390F


481
522
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390G


459
500
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390H


497
538
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390K


454
495
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390P


465
506
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390Q


480
521
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390R


504
545
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K/M390S


463
504
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
34




E367N/R373K/M390T


496
537
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K/M390V


491
532
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M390W


457
498
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
34




E367N/R373K/M392A


483
524
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
34




E367N/R373K/M392C


455
496
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
35




E367N/R373K/M392D


466
507
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
35




E367N/R373K/M392E


479
520
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M392F


501
542
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M392G


498
539
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
34




E367N/R373K/M392I


472
513
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
35




E367N/R373K/M392K


473
514
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
33




E367N/R373K/M392L


505
546
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M392N


493
534
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M392P


461
502
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
35




E367N/R373K/M392Q


478
519
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K/M392S


507
548
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M392T


484
525
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
34




E367N/R373K/M392V


485
526
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/M392W


503
544
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/Q385C


482
523
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/Q385G


469
510
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K/Q385I


462
503
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K/Q385L


509
550
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/Q385T


506
547
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/Q385W


514
555
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
31




E367N/R373K/S393*


492
533
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K/T389C


475
516
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/T389D


487
528
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/T389G


489
530
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K/T389I


499
540
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
35




E367N/R373K/T389L


456
497
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
35




E367N/R373K/T389M


488
529
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/T389N


495
536
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
34




E367N/R373K/T389P


468
509
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/T389Q


467
508
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/T389S


471
512
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
30




E367N/R373K/T389W


327
368
L44R/K206A/F217R/N247D/Q302K/L316D/M322I/K343D/A350G/
427
37




K362Q/E367N/R373K


434
475
L44R/K206A/F217R/N247D/R270D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


431
472
L44R/K206A/F217R/N247D/R270G/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


453
494
L44R/K206A/F217R/N247D/R270L/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


448
489
L44R/K206A/F217R/N247D/R270Q/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


280
321
L44R/K206A/F217R/Q302K/E367D/T369D
426
37


328
369
L44R/K206A/F217R/Q302K/M322I/A337P/A350G/E367N/T369S/
452
42




R373K


329
370
L44R/K206A/I208V/N247D/Q302K/M322I/K343D/E367N/R373K
442
38


330
371
L44R/K206A/I208V/R221K/M322I/K343D/E367N/R373K
443
38


281
322
L44R/K206A/I208V/R221K/M322V/K343D/F365L/R373K
422
37


331
372
L44R/K206A/I208V/R221K/N247D/Q302K/M322I/K343D/E367N/
441
38




R373K/I376V


332
373
L44R/K206A/I208V/R221T/Q302K/M322I/K343G/F365L/E367N/
449
40




R373K/I376V


333
374
L44R/K206A/L316D/M322I/A337P/A350G/E367N/T369S/R373K
450
42


282
323
L44R/K206A/N247D/A337P
429
38


334
375
L44R/K206A/N247D/L316D/M322I/A350G/K362Q/E367N/T369S/
443
42




R373K


283
324
L44R/K206A/N247D/Q302K/A337P/A350G/E367D/T369D
419
37


335
376
L44R/K206A/N247D/Q302K/L316D/M322I/A337P/K343G/A350G/
432
42




K362Q/E367N/T369S/R373K


336
377
L44R/K206A/N247D/Q302K/M322I/A350G/E367N/T369S/R373K
457
42


337
378
L44R/K206A/N247D/Q302K/M322I/K343D/E367N/R373K
442
38


284
325
L44R/K206A/R221T/N247D/M322I/K343D/F365L/I376V
432
37


285
326
L44R/K96I/K206A
419
36


286
327
L44R/K96I/K206A/F217R/N247D
418
36


338
379
L44R/K96I/K206A/F217R/N247D/L316D/M322I/A337P/A350G/
410
35




K362Q/E367N/R373K


339
380
L44R/K96I/K206A/F217R/N247D/M322I/A350G/K362Q/E367N/
418
35




R373K


340
381
L44R/K96I/K206A/F217R/N247D/M322I/A350G/K362Q/E367N/
428
40




T369S/R373K


341
382
L44R/K96I/K206A/F217R/N247D/M322I/E367N/T369S/R373K
440
40


287
328
L44R/K96I/K206A/F217R/N247D/Q302K/A337P/A350G
412
36


288
329
L44R/K96I/K206A/F217R/N247D/Q302K/A337P/K343D/A350G/
397
35




E367D/T369D


342
383
L44R/K96I/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
423
36




E367N/R373K


343
384
L44R/K96I/K206A/F217R/N247D/Q302K/M322I/E367N/T369S/
440
40




R373K


344
385
L44R/K96I/K206A/F217R/N247D/Q302K/M322I/K362Q/E367N/
418
35




R373K


345
386
L44R/K96I/K206A/F217R/Q219P/N247D/M253K/S266F/D284E/
429
41




Q290P/L293F/Q302K/V308G/S314F/M322I/A337P/K343E/E367N/




R373K


289
330
L44R/K96I/K206A/F217R/Q302K/A350G
419
36


346
387
L44R/K96I/K206A/F217R/Q302K/M322I/A350G/K362Q/E367N/
429
40




T369S/R373K


347
388
L44R/K96I/K206A/M322I/A337P/E367N/T369S/R373K
435
40


290
331
L44R/K96I/K206A/N247D/L316D/A337P/A350G/E367D/T369D
400
35


291
332
L44R/L100F/K206A/F365L
426
38


292
333
L44R/L100F/K206A/I208V/Q219H/N247D/Q302K/M322V/K343D/
416
37




R373K/I376V


348
389
L44R/L100F/K206A/I208V/R221K/M322I/K343G/F365L/E367N/
442
40




R373K


293
334
L44R/L100F/K206A/I208V/R221K/N247D/Q302K/M322V/F365L/
418
37




I376V


349
390
L44R/L100F/K206A/I208V/R221T/N247D/M322I/F365L/E367N/
446
40




R373K


350
391
L44R/L100F/K206A/I208V/R221T/N247D/M322V/E367N/R373K/
427
38




I376V


294
335
L44R/L100F/K206A/I208V/R221T/N247D/M322V/I376V
420
37


295
336
L44R/L100F/K206A/I208V/R221T/N247D/Q302K/M322I/K343D/
424
37




F365L/R373K/I376V


351
392
L44R/L100F/K206A/I208V/R221T/Q302K/M322I/E367N/R373K/
440
38




I376V


352
393
L44R/L100F/K206A/Q302K/M322I/E367N/R373K/I376V
440
38


353
394
L44R/L100F/K206A/R221K/M322I/F365L/E367N/R373K/I376V
446
40


354
395
L44R/L100F/K206A/R221T/M322I/F365L/E367N/R373K
447
40


355
396
L44R/L100F/K206A/R221T/N247D/M322I/K343D/E367N/R373K/
433
38




I376V


356
397
L44R/L100F/K206A/R221T/N247D/Q302K/M322I/E367N/R373K
440
38


357
398
L44R/L100F/K206A/R221T/N247D/Q302K/M322V/E367N/R373K/
427
38




I376V


358
399
L44R/L100F/K206A/R221T/Q302K/M322I/E367N/R373K
441
38


359
400
L44R/L100F/Q181L/K206A/R221T/N247D/Q302K/M322V/E367N/
429
38




R373K/I376V


400
441
L44R/L158C/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


405
446
L44R/L158E/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


422
463
L44R/L158G/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


407
448
L44R/L158H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


396
437
L44R/L158M/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
39




K362Q/E367N/R373K


414
455
L44R/L158Q/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


397
438
L44R/L158R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


121
165
L44R/L384F
455
35


418
459
L44R/N161E/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


523
564
L44R/N91M/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
396
28




A337P/K362Q/E367N/R373K


524
565
L44R/N91V/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
398
27




A337P/K362Q/E367N/R373K


525
566
L44R/Q76H/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
388
23




A337P/K362Q/E367N/W368A/R373K


423
464
L44R/R162A/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
35




K362Q/E367N/R373K


416
457
L44R/R162G/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


410
451
L44R/R162H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


406
447
L44R/R162K/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


421
462
L44R/R162Q/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


417
458
L44R/R162S/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


409
450
L44R/R165H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


399
440
L44R/R165K/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


526
567
L44R/R74H/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


411
452
L44R/S166A/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


415
456
L44R/S166D/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


419
460
L44R/S166E/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


404
445
L44R/S166F/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


403
444
L44R/S166G/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
35




K362Q/E367N/R373K


412
453
L44R/S166H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


402
42
L44R/S166P/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


408
449
L44R/S166R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


420
461
L44R/S166T/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


363
404
L44R/S47D/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


375
416
L44R/S47I/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


365
406
L44R/S47N/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


623
664
L44R/S47N/S166P/K206A/F217R/N247D/H271A/A276S/Q302K/
386
25




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


624
665
L44R/S47N/S166P/K206A/F217R/N247D/H271A/Q302K/L316D/
385
25




M322I/A337P/K362Q/E367N/R373K/M390Q


628
668
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
350
12




L316D/M322I/A337P/K362Q/E367N/R373K/M390H


613
654
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
351
12




L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


632
672
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
352
12




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


627
667
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/M259W/H271A/
311
5




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390H/M392T


622
663
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/M259W/H271A/
305
5




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q/M392T


615
656
L44R/S47N/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/
352
13




M322I/A337P/K362Q/E367N/R373K/M390H


361
402
L44R/S47R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


631
671
L44R/S47T/A53S/Y92H/S166P/K206A/F217R/N247D/H271A/
344
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


379
420
L44R/S47T/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
32




K362Q/E367N/R373K


675
715
L44R/S47T/M65V/Y92H/S166P/K206A/F217R/N247D/H271A/
344
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


657
697
L44R/S47T/P67T/Y92H/S166P/K182N/K206A/F217R/N247D/
338
8




H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


629
669
L44R/S47T/S166P/K206A/F217R/N247D/H271A/Q302K/L316D/
378
21




M322I/A337P/K362Q/E367N/R373K/M390Q


659
699
L44R/S47T/W64L/Y92H/S166P/K206A/F217R/N247D/H271A/
345
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


672
712
L44R/S47T/Y92H/D144Y/S166P/K206A/F217R/N247D/H271A/
351
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


646
686
L44R/S47T/Y92H/G113C/S166P/K206A/F217R/N247D/H271A/
345
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


644
684
L44R/S47T/Y92H/S166P/K206A/F217R/L237P/N247D/H271A/
338
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


687
727
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q290R/
340
7




Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


618
659
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
361
15




L316D/M322I/A337P/K362Q/E367N/R373K


619
660
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
343
8




L316D/M322I/A337P/K362Q/E367N/R373K/M390H


620
661
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
344
8




L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


625
44
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
345
8




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


673
713
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
331
7




L316D/M322I/A337P/K362Q/E367N/R373K/N377Y/M392T


693
733
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
340
7




L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


682
722
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
348
8




L316Y/M322I/A337P/K362Q/E367N/R373K/M392T


670
710
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
345
8




N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


689
729
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
345
8




N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


684
724
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
340
7




N305L/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


678
718
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M253W/A257G/
339
7




H271A/K277R/Q281L/Q302K/L316D/A319D/M322I/A337P/K362Q/




E367N/R373K/M392T


677
717
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M253W/H271A/
338
7




S273D/P274S/K277R/Q302K/L316D/M322I/A337P/K362Q/E367N/




R373K/M392T


621
662
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M259E/H271A/
307
1




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


610
651
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M259E/Q302K/
310
2




L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


630
670
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M259W/H271A/
311
1




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390H


616
657
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/M259W/H271A/
312
1




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


669
709
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/P262S/H271A/
353
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


614
655
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/
363
16




M322I/A337P/K362Q/E367N/R373K


692
732
L44R/S47T/Y92H/S166P/K206A/F217R/N247D/W256L/H271/
347
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


666
706
L44R/S47T/Y92H/S166P/K206A/F217R/P228L/N247D/H271A/
345
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


688
728
L44R/S47T/Y92H/S166P/K206A/F217R/P228Q/N247D/H271A/
345
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


674
714
L44R/S47T/Y92H/S166P/K206A/F217R/P234H/N247D/H271A/
345
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


662
702
L44R/S47T/Y92H/S166P/K206A/F217R/V238I/N247D/H271A/
347
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


652
692
L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/A261G/
312
1




H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/




M392T


655
695
L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/A261G/
307
0




H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/W368A/




R373K/M392T


645
685
L44R/S47T/Y92H/S166P/P174S/K206A/F217R/N247D/H271A/
340
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


661
701
L44R/S47T/Y92H/S166P/W195C/K206A/F217R/N247D/H271A/
345
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


366
407
L44R/S47V/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


401
442
L44R/T163S/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
34




K362Q/E367N/R373K


388
429
L44R/V93L/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


389
430
L44R/V93S/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
29




K362Q/E367N/R373K


387
428
L44R/V93T/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
31




K362Q/E367N/R373K


385
426
L44R/Y92A/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
25




K362Q/E367N/R373K


383
424
L44R/Y92C/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
24




K362Q/E367N/R373K


527
568
L44R/Y92E/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
377
24




K362Q/E367N/R373K


393
434
L44R/Y92G/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
24




K362Q/E367N/R373K


528
569
L44R/Y92H/D130Q/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


529
570
L44R/Y92H/K182A/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


530
571
L44R/Y92H/K182E/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


531
572
L44R/Y92H/K182H/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


532
573
L44R/Y92H/K182M/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


533
574
L44R/Y92H/K182Q/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


534
575
L44R/Y92H/K182R/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


535
576
L44R/Y92H/K182T/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


536
577
L44R/Y92H/K182V/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


537
578
L44R/Y92H/K182Y/K206A/F217R/N247D/Q302K/L316D/M322I/
386
24




A337P/K362Q/E367N/R373K


538
579
L44R/Y92H/K206A/F217R/N247D/A287C/Q302K/L316D/M322I/
392
24




A337P/K362Q/E367N/R373K


539
580
L44R/Y92H/K206A/F217R/N247D/A287H/Q302K/L316D/M322I/
394
24




A337P/K362Q/E367N/R373K


540
581
L44R/Y92H/K206A/F217R/N247D/A287M/Q302K/L316D/M322I/
404
24




A337P/K362Q/E367N/R373K


541
582
L44R/Y92H/K206A/F217R/N247D/K283A/Q302K/L316D/M322I/
384
24




A337P/K362Q/E367N/R373K


542
583
L44R/Y92H/K206A/F217R/N247D/K283G/Q302K/L316D/M322I/
387
24




A337P/K362Q/E367N/R373K


543
584
L44R/Y92H/K206A/F217R/N247D/K283M/Q302K/L316D/M322I/
385
24




A337P/K362Q/E367N/R373K


544
585
L44R/Y92H/K206A/F217R/N247D/K283V/Q302K/L316D/M322I/
385
24




A337P/K362Q/E367N/R373K


545
586
L44R/Y92H/K206A/F217R/N247D/K295A/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


546
587
L44R/Y92H/K206A/F217R/N247D/K295E/Q302K/L316D/M322I/
392
24




A337P/K362Q/E367N/R373K


547
588
L44R/Y92H/K206A/F217R/N247D/K295L/Q302K/L316D/M322I/
409
24




A337P/K362Q/E367N/R373K


548
589
L44R/Y92H/K206A/F217R/N247D/K295N/Q302K/L316D/M322I/
391
24




A337P/K362Q/E367N/R373K


549
590
L44R/Y92H/K206A/F217R/N247D/K295Q/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


550
591
L44R/Y92H/K206A/F217R/N247D/K295S/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


551
592
L44R/Y92H/K206A/F217R/N247D/K295T/Q302K/L316D/M322I/
392
24




A337P/K362Q/E367N/R373K


552
593
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/A317D/M322I/
393
24




A337P/K362Q/E367N/R373K


553
594
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/A317Q/M322I/
393
24




A337P/K362Q/E367N/R373K


554
595
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
388
24




A346G/K362Q/E367N/R373K


555
596
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
395
24




G344A/K362Q/E367N/R373K


556
597
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
388
24




G344D/K362Q/E367N/R373K


557
598
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
388
24




G344S/K362Q/E367N/R373K


558
599
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
391
24




I353L/K362Q/E367N/R373K


559
600
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
386
23




K362Q/E367N/L372W/R373K


395
40
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
24




K362Q/E367N/R373K


560
601
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
388
23




K362Q/E367N/W368A/R373K


561
602
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
412
31




K362Q/E367N/W368L/R373K


562
603
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
393
24




K362Q/E367N/W368N/R373K


563
604
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
400
28




K362Q/E367N/W368R/R373K


564
605
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
407
29




K362Q/E367N/W368V/R373K


565
606
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
393
24




N348E/K362Q/E367N/R373K


566
607
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
393
24




N348M/K362Q/E367N/R373K


567
608
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
393
24




N348Q/K362Q/E367N/R373K


568
609
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
393
24




N348R/K362Q/E367N/R373K


569
610
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
393
24




N348W/K362Q/E367N/R373K


570
611
L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
391
24




T354S/K362Q/E367N/R373K


571
612
L44R/Y92H/K206A/F217R/N247D/Q302K/N305K/L316D/M322I/
400
24




A337P/K362Q/E367N/R373K


572
613
L44R/Y92H/K206A/F217R/N247D/Q302K/N305L/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


573
614
L44R/Y92H/K206A/F217R/N247D/Q302K/S314A/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


574
615
L44R/Y92H/K206A/F217R/N247D/Q302K/S314H/L316D/M322I/
395
24




A337P/K362Q/E367N/R373K


575
616
L44R/Y92H/K206A/F217R/N247D/Q302K/S314N/L316D/M322I/
388
24




A337P/K362Q/E367N/R373K


576
617
L44R/Y92H/K206A/F217R/N247D/Q302K/S314Y/L316D/M322I/
388
24




A337P/K362Q/E367N/R373K


577
618
L44R/Y92H/K206A/F217R/W246A/N247D/Q302K/L316D/M322I/
395
24




A337P/K362Q/E367N/R373K


578
619
L44R/Y92H/K206A/F217R/W246I/N247D/Q302K/L316D/M322I/
399
24




A337P/K362Q/E367N/R373K


579
620
L44R/Y92H/K206A/F217R/W246P/N247D/Q302K/L316D/M322I/
387
24




A337P/K362Q/E367N/R373K


580
621
L44R/Y92H/K206A/F217R/W246R/N247D/Q302K/L316D/M322I/
396
24




A337P/K362Q/E367N/R373K


581
622
L44R/Y92H/K206A/F217R/W246S/N247D/Q302K/L316D/M322I/
402
24




A337P/K362Q/E367N/R373K


582
623
L44R/Y92H/K206A/S210A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/A350T/K362Q/E367N/R373K


583
624
L44R/Y92H/K206A/S210A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


584
625
L44R/Y92H/K206A/S210E/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


585
626
L44R/Y92H/K206A/S210K/F217R/N247D/Q302K/L316D/M322I/
407
24




A337P/K362Q/E367N/R373K


586
627
L44R/Y92H/K206A/S210N/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


587
628
L44R/Y92H/K206A/S210R/F217R/N247D/Q302K/L316D/M322I/
408
24




A337P/K362Q/E367N/R373K


588
629
L44R/Y92H/K96A/K206A/F217R/N247D/Q302K/L316D/M322I/
380
24




A337P/K362Q/E367N/R373K


589
630
L44R/Y92H/K96W/K206A/F217R/N247D/Q302K/L316D/M322I/
399
26




A337P/K362Q/E367N/R373K


617
658
L44R/Y92H/L136V/S166P/K206A/F217R/N247D/M259A/Q302K/
347
8




L316D/M322I/A337P/K362Q/E367N/R373K/M390Q


590
631
L44R/Y92H/P179M/K206A/F217R/N247D/Q302K/L316D/M322I/
414
29




A337P/K362Q/E367N/R373K


591
632
L44R/Y92H/R189K/K206A/F217R/N247D/Q302K/L316D/M322I/
390
24




A337P/K362Q/E367N/R373K


592
633
L44R/Y92H/R189V/K206A/F217R/N247D/Q302K/L316D/M322I/
398
24




A337P/K362Q/E367N/R373K


626
666
L44R/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/L316D/
358
13




M322I/A337P/K362Q/E367N/R373K/M390Q


611
652
L44R/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/M322I/
360
14




A337P/K362Q/E367N/R373K/M390Q


612
953
L44R/Y92H/S166P/K206A/F217R/N247D/Q302K/L316D/M322I/
361
14




A337P/K362Q/E367N/R373K/M392T


593
634
L44R/Y92H/S95A/K206A/F217R/N247D/Q302K/L316D/M322I/
396
27




A337P/K362Q/E367N/R373K


594
635
L44R/Y92H/S95E/K206A/F217R/N247D/Q302K/L316D/M322I/
375
24




A337P/K362Q/E367N/R373K


595
636
L44R/Y92H/T186A/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


596
637
L44R/Y92H/T186G/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


597
638
L44R/Y92H/T186V/K206A/F217R/N247D/Q302K/L316D/M322I/
401
24




A337P/K362Q/E367N/R373K


598
639
L44R/Y92H/Y120H/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


599
640
L44R/Y92H/Y120S/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


600
641
L44R/Y92H/Y120S/K206A/F217R/N247D/Q302K/L316D/M322I/
388
24




A337P/L341F/K362Q/E367N/R373K


380
421
L44R/Y92K/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
24




K362Q/E367N/R373K


390
431
L44R/Y92Q/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
24




K362Q/E367N/R373K


394
435
L44R/Y92R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
30




K362Q/E367N/R373K


381
422
L44R/Y92S/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
24




K362Q/E367N/R373K


392
433
L44R/Y92T/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
24




K362Q/E367N/R373K


384
425
L44R/Y92V/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
35




K362Q/E367N/R373K


369
410
L44S/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K


122
166
L44T
456
37


378
419
L44T/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K


372
413
L44V/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
36




E367N/R373K


371
412
L44W/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/K362Q/
N.D.
32




E367N/R373K


123
167
M20D/Q302K
450
38


124
168
M253F
444
38


125
169
M322I
462
38


126
170
M390D
425
31


127
171
M390R
430
31


128
172
M390T
438
35


129
173
M392G
435
31


130
174
M392P
433
31


131
175
M392S
448
37


601
642
M39C/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
367
19




A337P/K362Q/E367N/R373K


683
723
M39E/E43D/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/
309
6




M253W/H271A/S273D/Q302K/L316D/M322I/A337P/K362Q/E367N/




W368A/R373K/M392T


660
700
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/H271A/
302
1




Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


668
708
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
329
8




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


676
716
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
324
7




L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


639
679
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247Y/H271A/Q302K/
343
8




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


658
698
M39E/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/H271A/
323
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


602
643
M39E/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
363
19




K362Q/E367N/R373K


364
405
M39H/L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
32




K362Q/E367N/R373K


367
408
M39R/L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
32




K362Q/E367N/R373K


603
644
M39R/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
368
19




A337P/K362Q/E367N/R373K


377
418
M39T/L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
32




K362Q/E367N/R373K


604
645
M39V/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/
393
24




A337P/K362Q/E367N/R373K


132
176
M39Y
451
37


376
417
M41P/L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
35




K362Q/E367N/R373K


373
414
M41R/L44R/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
N.D.
36




K362Q/E367N/R373K


133
177
N388R
454
38


134
178
N91Q
438
32


26
72
P179S/R373K
430
37


135
179
Q190S/T369D
448
38


136
180
Q249A
449
38


27
73
Q299R/M322V/R373K
451
37


28
74
Q299R/Q302K/R373K
451
37


29
75
Q299R/Q302K/R373K/I376V
450
37


137
181
Q302A
450
38


30
76
Q302K/I376V
443
37


138
182
Q385H
435
32


139
183
Q385I
447
38


140
184
Q385L
445
38


141
185
Q391G
449
36


142
186
Q80A
450
38


143
187
Q80H
450
38


144
188
Q80V
459
38


145
189
Q88A
448
38


146
190
Q88F
456
38


147
191
Q88H
448
38


148
192
Q88R
448
38


149
193
Q88S
448
38


150
194
R162H
446
35


151
195
R162S
450
37


225
226
R165S/K206A
427
39


152
196
R221K/A350G
450
38


153
197
R221T
450
38


154
198
R301I/K362T
449
41


155
199
R301L
450
38


156
200
R371S
456
39


157
201
R371V
452
40


31
77
R373K
444
37


32
78
R373K/I376V
443
37


665
705
R7C/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
340
7




L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


650
690
R7H/T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
345
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


681
721
R7P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
345
8




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


654
694
R7S/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
345
8




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


158
202
R87K
435
32


159
203
R87P/L398R
423
31


160
204
S166A
440
35


161
205
S166H
447
35


162
206
S166K
441
35


163
207
S31D
450
38


164
208
S34D/M392P
439
31


165
209
S34G
450
38


166
210
S34H/M390R
430
31


167
211
S34R
450
38


168
212
S374M
454
40


169
213
S374T
439
37


170
214
S393E
447
37


171
215
S393G
447
37


172
216
S393H
454
38


173
217
S393P
452
37


174
218
S47I
450
38


175
219
S47R
459
38


176
220
S47T
433
33


177
221
S95D
422
31


178
222
S95E
414
31


179
223
S95Q
446
36


686
728
T10P/E17G/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
352
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


642
682
T10P/E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/
315
1




H271A/Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/W368A/




R373K/M392T


690
730
T10P/L44R/S47T/Y92H/M156V/S166P/K206A/F217R/N247D/H271A/
333
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


638
678
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/H271A/
318
1




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


651
691
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
345
8




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


691
731
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
345
8




L316D/M322I/A337P/K362Q/E367N/R373K/M392T


671
711
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
340
7




L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


643
683
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/Q302K/
335
8




L316D/M322I/R325S/A337P/K362Q/E367N/R373K/M392T


664
704
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/Q252H/M253R/
312
2




A254E/A261G/H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/




R373K/M392T


656
696
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/A261G/
312
1




H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


653
693
T10P/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/H271A/
339
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


605
646
T10P/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
393
24




K362Q/E367N/R373K


606
647
T10P/L44R/Y92H/R189L/K206A/F217R/N247D/Q302K/L316D/M322I/
395
24




A337P/K362Q/E367N/R373K


640
680
T10P/M39E/E43D/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/
329
8




H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


648
46
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/A261G/
297
0




H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/W368A/R373K/M392T


680
720
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
329
8




Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


679
719
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/H271A/
329
8




Q302K/N305L/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


641
681
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/N247D/S266P/
313
8




H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


649
689
T10P/M39E/L44R/S47T/Y92H/S166P/K206A/F217R/W246P/N247D/
323
8




H271A/Q302K/L316D/M322I/A337P/K362Q/E367N/R373K/M392T


180
224
T369D
447
38


181
225
T369S
450
38


182
226
T389S
436
31


607
648
T8L/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
398
24




K362Q/E367N/R373K


608
649
T8Q/L44R/Y92H/K206A/F217R/N247D/Q302K/L316D/M322I/A337P/
393
24




K362Q/E367N/R373K


183
227
V133I
457
38


184
228
V168A
434
37


185
229
V168L
445
38


186
230
V345N
447
38


187
231
V345Y
449
38


188
232
V359E
429
38


189
233
V93I
443
37


190
234
W178H
448
38


191
235
W178S
442
38





N.D.—Not determined.






While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.


For all purposes in the United States of America, each and every publication and patent document cited in this application is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date.

Claims
  • 1. A recombinant alpha galactosidase A comprising an amino acid sequence comprising at least 95%, sequence identity to SEQ ID NO: 5, wherein said amino acid sequence comprises a mutation at position 206, wherein said positions are numbered with reference to SEQ ID NO: 5.
  • 2. The recombinant alpha galactosidase A of claim 1, wherein said recombinant alpha galactosidase A is a recombinant human alpha galactosidase A.
  • 3. The recombinant alpha galactosidase A of claim 1, wherein said recombinant alpha galactosidase A exhibits at least one improved property selected from: i) increased thermostability; ii) enhanced catalytic activity; iii) increased tolerance to pH 7.4; iv) increased tolerance to pH 4.3; v) increased tolerance to serum; or vi) reduced immunogenicity; or a combination of any of i), ii), iii), iv), v), or vi), as compared to a reference alpha galactosidase A.
  • 4. The recombinant alpha galactosidase A of claim 3, wherein said recombinant alpha galactosidase A is more thermostable than the alpha galactosidase A of SEQ ID NO:5.
  • 5. The recombinant alpha galactosidase A of claim 3, wherein said recombinant alpha galactosidase A is more stable at pH 7.4 than the alpha galactosidase A of SEQ ID NO:5.
  • 6. The recombinant alpha galactosidase A of claim 3, wherein said recombinant alpha galactosidase A is more stable at pH 4.3 than the alpha galactosidase A of SEQ ID NO:5.
  • 7. The recombinant alpha galactosidase A of claim 3, wherein said recombinant alpha galactosidase A is more stable to exposure to serum than the alpha galactosidase A of SEQ ID NO:5.
  • 8. The recombinant alpha galactosidase A of claim 3, wherein said recombinant alpha galactosidase A is less immunogenic than wild-type alpha galactosidase A.
  • 9. The recombinant alpha galactosidase A of claim 1, further comprising at least one mutation at a position selected from position 2, 7, 8, 10, 14, 15, 17, 20, 21, 23, 24, 30, 31, 34, 36, 37, 39, 40, 41, 43, 44, 47, 48, 52, 53, 56, 59, 64, 65, 66, 67, 74, 76, 77, 80, 84, 87, 88, 91, 92, 93, 94, 95, 96, 100, 102, 105, 113, 120, 123, 124, 125, 130, 133, 136, 143, 144, 147, 155, 156, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 169, 170, 174, 177, 178, 179, 180, 181, 182, 186, 187, 189, 190, 195, 198, 199, 208, 210, 217, 219, 221, 228, 230, 234, 237, 238, 246, 247, 249, 252, 253, 254, 255, 256, 257, 258, 259, 261, 262, 263, 266, 269, 270, 271, 273, 274, 276, 277, 281, 283, 284, 287, 290, 293, 295, 299, 301, 302, 303, 305, 308, 314, 316, 317, 319, 322, 325, 326, 337, 339, 343, 344, 345, 346, 348, 349, 350, 352, 353, 354, 359, 362, 365, 367, 368, 369, 371, 373, 374, 375, 376, 377, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, and 398, wherein the positions are numbered relative to SEQ ID NO: 5.
  • 10. The recombinant alpha galactosidase A of claim 1, wherein said recombinant alpha galactosidase A is purified.
  • 11. The recombinant alpha galactosidase A of claim 10, wherein said reference sequence is selected from wild-type alpha galactosidase A, SEQ ID NO:5, and SEQ ID NO:10.
  • 12. A composition comprising said recombinant alpha galactosidase A of claim 1.
  • 13. A pharmaceutical composition for the treatment of Fabry disease, comprising the enzyme composition of claim 12, further comprising a pharmaceutically acceptable carrier and/or excipient.
  • 14. The pharmaceutical composition of claim 13, wherein said composition is suitable for parenteral injection or infusion to a human.
  • 15. Use of the composition of claim 12.
Parent Case Info

The present application is a Continuation application that claims priority to co-pending U.S. patent application Ser. No. 15/529,383, filed May 24, 2017, which is a national stage application filed under 35 USC § 371 and claims priority to PCT International Application No. PCT/US2015/063329, filed Dec. 2, 2015, which claims priority to U.S. Prov. Pat. Application Ser. No. 62/095,313, filed Dec. 22, 2014, and U.S. Prov. Pat. Application Ser. No. 62/216,452, filed Sep. 10, 2015, all of which are hereby incorporated by reference in their entireties for all purposes.

Provisional Applications (2)
Number Date Country
62095313 Dec 2014 US
62216452 Sep 2015 US
Continuations (1)
Number Date Country
Parent 15529383 May 2017 US
Child 16985742 US