MU-DIGUETOXIN-DC1A VARIANT POLYPEPTIDES FOR PEST CONTROL

SEQUENCE LISTING

This application incorporates by reference in its entirety the Sequence Listing entitled “225312-497884_ST25.txt” (126 kilobytes), which was created on Sep. 27, 2021, at 6:32 PM, that is 126 KB, and filed electronically herewith.

TECHNICAL FIELD

The present disclosure provides insecticidal proteins, nucleotides, peptides, their expression in plants, methods of producing the peptides, new formulations, and methods for the control of insects are described.

BACKGROUND

Deleterious insects represent a worldwide threat to human health and food security. Insects pose a threat to human health because they are a vector for disease. One of the most notorious insect-vectors of disease is the mosquito. Mosquitoes in the genus Anopheles are the principal vectors of Zika virus, Chikungunya virus, and malaria—a disease caused by protozoa in the genus Trypanosoma. Another mosquito, Aedes aegypti, is the main vector of the viruses that cause Yellow fever and Dengue. And, Aedes spp. mosquitos are also the vectors for the viruses responsible for various types of encephalitis. Wuchereria bancrofti and Brugia malayi, parasitic roundworms that cause filariasis, are usually spread by mosquitoes in the genera Culex, Mansonia, and Anopheles.

Similar to the mosquito, other members of the Diptera order have likewise plagued humankind since time immemorial. In addition to producing painful bites, Horseflies and deerflies transmit the bacterial pathogens of tularemia (Pasteurella tularensis) and anthrax (Bacillus anthracis), as well as a parasitic roundworm (Loa loa) that causes loiasis in tropical Africa.

Blowflies (Chrysomya megacephala) and houseflies (Musca domestica) will in one moment take off from carrion and dung, and in the next moment alight in our homes and on our food-spreading dysentery, typhoid fever, cholera, poliomyelitis, yaws, leprosy, and tuberculosis in their wake.

Eye gnats in the genus Hippelates can carry the spirochaete pathogen that causes yaws (Treponema pertenue), and may also spread conjunctivitis (pinkeye). Tsetse flies in the genus Glossina transmit the protozoan pathogens that cause African sleeping sickness (Trypanosoma gambiense and T. rhodesiense). Sand flies in the genus Phlebotomus are vectors of a bacterium (Bartonella bacilliformis) that causes Carrion's disease (Oroyo fever) in South America. In parts of Asia and North Africa, they spread a viral agent that causes sand fly fever (Pappataci fever) as well as protozoan pathogens (Leishmania spp.) that cause Leishmaniasis.

Human food security is also threatened by insects. Insect pests indiscriminately target food crops earmarked for commercial purposes and personal use alike; indeed, the damage caused by insect pests can run the gamut from mere inconvenience to financial ruin in the former, to extremes such as malnutrition or starvation in the latter. Insect pests also cause stress and disease in domesticated animals. And, insect pests once limited by geographical and climate boundaries have expanded their range due to global travel and climate change.

SUMMARY

The present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species. Here, the DVP comprises an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K-K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁₁-X₁₂-C-R-D-V, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X₁₁is C, F, A, T, S, M, or V; and X₁₂is V, A, or T; or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a composition consisting of a DVP, a DVP-insecticidal protein, or combinations thereof, and an excipient.

The present disclosure describes a polynucleotide operable to encode a DVP, where the DVP comprises an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C—K-K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F—F-S-S-K-X₁₁-X₁₂-C-R-D-V, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X₁₁is C, F, A, T, S, M, or V; and X₁₂is V, A, or T, or a complementary nucleotide sequence thereof.

In addition, the present disclosure describes a method of producing a DVP, the method comprising: preparing a vector comprising a first expression cassette comprising a polynucleotide operable to encode a DVP, and/or a complementary nucleotide sequence thereof, said DVP comprising an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K—K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁₁—X₁₂-C-R-D-V, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X₁₁is C, F, A, T, S, M, or V; and X₁₂is V, A, or T; introducing the vector into a yeast cell; and growing the yeast cell in a growth medium under conditions operable to enable expression of the DVP and secretion into the growth medium.

The present disclosure describes a method of combating, controlling, or inhibiting a pest comprising, applying a pesticidally effective amount of the composition consisting of a DVP, a DVP-insecticidal protein, or combinations thereof, and an excipient, to the locus of the pest, or to a plant or animal susceptible to an attack by the pest.

In addition, the present disclosure describes a vector comprising a polynucleotide operable to encode a DVP having an amino sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191,202-215, or 217-219.

The present disclosure also describes a yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode a DVP, said DVP comprising an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K-K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁₁—X₁₂-C-R-D-V, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X₁₁is C, F, A, T, S, M, or V; and X₁₂is V, A, or T.

In addition, the present disclosure provides a recombinant CRP comprising, consisting essentially of, or consisting of, a cystine knot (CK) architecture according to Formula (II):

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In addition, the present disclosure describes a method of making a recombinant cysteine-rich protein (CRP) comprising a cystine knot (CK) architecture according to Formula (II):

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The present disclosure also describes a method of increasing the yield of a recombinant cysteine-rich protein (CRP), said method comprising: (a) creating a recombinant CRP having a cystine knot (CK) architecture according to Formula (II):

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In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP comprising an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP consisting of an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP consisting of an amino acid set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP comprising an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP consisting of an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP consisting of an amino acid set forth in any one of SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP comprising an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP consisting of an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP consisting of an amino acid set forth in any one of SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP comprising an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 213, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a diguetoxin variant polypeptide (DVP) having insecticidal activity against one or more insect species, said DVP consisting of an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 213, or 217-219, or a pharmaceutically acceptable salt thereof.

In addition, the present disclosure describes a fusion protein comprising one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein said one or more DVPs have an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K—K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁₁-X₁₂-C-R-D-V, wherein the DVP comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X₁₁is C, F, A, T, S, M, or V; and X₁₂is V, A, or T, or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the high-performance liquid chromatography (HPLC) standard curve for wild-type (WT) Dc1a.

FIG. 2 shows an HPLC chromatogram for pure WT Dc1a.

FIG. 3 depicts a graph showing the relative yield of DVPs C41T/C51A and C41T/C51A/W31F/Y32S/P36A. The DVP C41T/C51A/W31F/Y32S/P36A had a 69% increase in expression compared to C41T/C51A.

FIG. 4 depicts a chromatogram of C41T/C51A. Peaks indicating the background, folded, and misfolded variants are shown in brackets.

FIG. 5 depicts a chromatogram of C41T/C51A/D38A/L42V. Peaks indicating the background, and folded variants are indicated by labels.

FIG. 6 depicts a graph showing a summary of the relative expression of DVPs, showing increased expression without loss of activity. Here, WT-Dc1a, and the following DVPs were analyzed: (1) C41T/C51A; (2) C41T/C51A/D38A; (3) C41T/C51A/D38A/L42V; and (4) C41S/C51S/D38A/L42V.

FIG. 7 shows the results of a fly knockdown experiment evaluating the effect of WT-Dc1a and the following DVPs: (1) C41T/C51A; (2) C41T/C51A/D38A; and (3) C41S/C51S/D38A/L42V. Dose-response curves were generated by assessing flies for percent knockdown (i.e., the inability to walk) at 24 hours (% Knockdown at 24 hr).

FIG. 8 depicts a graph showing percent knockdown for wild-type (triangle), and the DVPs: (1) C41T/C51A/D38A (SEQ ID NO:29) (diamond) and C41S/C51S/D38A/L42V (SEQ ID NO:53) (square), at 24 hours.

FIG. 9 depicts a schematic of a DVP-insecticidal protein. Here, the components are defined as follows: “ERSP” refers to the endoplasmic reticulum signal peptide; “UBI” refers to a ubiquitin monomer; “DVP” refers to a Mu-diguetoxin variant polypeptide; “L” refers to intervening linker peptide; and “HIS” refers to Histidine tag.

FIG. 10 depicts a His-Tag western blot of plant expressed WT Dc1a and DVP-insecticidal proteins. Each lane represents crude plant extracts run under denaturing protein gel conditions and visualized with standard western blot techniques. The short name for the samples tested in the western blot are listed above the image along with a rating system for expression. The symbol (−) indicates that there is no protein detected on the blot and if protein is detected, the symbol (+) to (+++) indicate the amount detected. The lane indicated “LADDER” shows the molecular weight marker. Lanes “PLANT NEG” show the negative control (i.e., GFP expressing tobacco protein extract). Lanes labeled with “M #” indicate the short name for the DVP-insecticidal protein evaluated. Lane “WT” shows an insecticidal protein having the WT Mu-diguetoxin-Dc1a protein.

FIG. 11 shows a graph demonstrating the yield of high yield DVPs compared to a background DVP. Here, point mutations were made on a background DVP having the following mutations: D38A, C41S, and C51S. Mutations to the background DVP included: L42I; K2L; Y32S; K2L+Y32S; D38T; D38S; and D38M. Yield was assessed via rpHPLC and normalized to the background DVP. DVPs with the additional mutations L42I; K2L; Y32S; K2L+Y32S; D38T; and D38S; all possessed improved yield relative to the C41S/C51S/D38A DVP background (SEQ ID NO: 47) control.

FIG. 12 shows a graph showing the result of K2L, Y32S, and L42I mutations. Here, the yield of the DVPs: (1) K2L/Y32S/L42I (SEQ ID NO: 217); and (2) K2L/Y32S/D38A/L42I/C41S/C51S (SEQ ID NO: 218); were compared to the yield of WT Dc1a (SEQ ID NO: 2). Combining the mutations K2L, Y32S, and L42I resulted in dramatic increases in the level of expression.

FIG. 13 depicts a schematic showing Formula (II), which describes a recombinant cysteine rich protein (CRP) having a cystine knot (CK) architecture. Here, C^Ito C^VIare cysteine residues; cysteine residues C^Iand C^IVare connected by a first disulfide bond; C^IIand C^Vare connected by a second disulfide bond; and C^IIIand C^VIare connected by a third disulfide bond; (disulfide bonds are indicated by lines connecting cysteine residues). The first disulfide bond, the second disulfide bond, and the third disulfide bond have a disulfide bond topology that forms a cystine knot motif, wherein the first disulfide bond, second disulfide bond, and third disulfide bond are the only disulfide bonds that form the cystine knot motif. N_E, L₁, L₂, L₃, L₄, L₅, and C_Eare peptide subunits each comprising an amino acid sequence having a length of 1 to 13 amino acid residues. In some embodiments, wherein N_E, L₃, C_E, or any combination thereof, are optionally absent.

FIG. 14 shows the relative yield of WT ApsIII and ApsIII cysteine deletion (dCys) as determined by HPLC. (n=8). The dashed line shows the median; dotted lines show the boundaries of the interquartile ranges.

DETAILED DESCRIPTION
Definitions

The term “5′-end” and “3′-end” refers to the directionality, i.e., the end-to-end orientation of a nucleotide polymer (e.g., DNA). The 5′-end of a polynucleotide is the end of the polynucleotide that has the fifth carbon.

“5′- and 3′-homology arms” or “5′ and 3′ arms” or “left and right arms” refers to the polynucleotide sequences in a vector and/or targeting vector that homologously recombine with the target genome sequence and/or endogenous gene of interest in the host organism in order to achieve successful genetic modification of the host organism's chromosomal locus.

“ACTX” or “ACTX peptide” or “atracotoxin” refers to a family of insecticidal ICK peptides that have been isolated from spiders belonging to the Atracinae family. One such spider is known as the Australian Blue Mountains Funnel-web Spider, which has the scientific name Hadronyche versuta. Examples of ACTX peptides from Atracinae family species are the Omega-ACTX, Kappa-ACTX, and U-ACTX peptides.

“ADN1 promoter” refers to the DNA segment comprised of the promoter sequence derived from the Schizosaccharomyces pombe adhesion defective protein 1 gene.

“Affect” refers to how a something influences another thing, e.g., how a peptide, polypeptide, protein, drug, or chemical influences an insect, e.g., a pest.

“Alignment” refers to a method of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide, or protein sequences) for the purpose of determining their relationship to each other. Alignments are typically performed by computer programs that apply various algorithms, however, it is also possible to perform an alignment by hand. Alignment programs typically iterate through potential alignments of sequences and score the alignments using substitution tables, employing a variety of strategies to reach a potential optimal alignment score. Commonly-used alignment algorithms include, but are not limited to, CLUSTALW (see Thompson J. D., Higgins D. G., Gibson T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research 22: 4673-4680, 1994); CLUSTALV (see Larkin M. A., et al., CLUSTALW2, ClustalW and ClustalX version 2, Bioinformatics 23(21): 2947-2948, 2007); Mafft; Kalign; ProbCons; and T-Coffee (see Notredame et al., T-Coffee: A novel method for multiple sequence alignments, Journal of Molecular Biology 302: 205-217, 2000). Exemplary programs that implement one or more of the foregoing algorithms include, but are not limited to, MegAlign from DNAStar (DNAStar, Inc. 3801 Regent St. Madison, Wis. 53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio from Accelrys (Accelrys, Inc., 10188 Telesis Ct, Suite 100, San Diego, Calif 92121). In some embodiments, an alignment will introduce “phase shifts” and/or “gaps” into one or both of the sequences being compared in order to maximize the similarity between the two sequences, and scoring refers to the process of quantitatively expressing the relatedness of the aligned sequences.

“Alpha mating factor (alpha-MF) peptide” or “alpha-MF signal” or “alpha-MF” or “alpha mating factor secretion signal” or “αMF secretion signal” (all used interchangeably) refers to a signal peptide that allows for secreted expression in a recombinant expression system, when the alpha-MF peptide is operably linked to a recombinant peptide of interest (e.g., a DVP). The Alpha-MF peptide directs nascent recombinant polypeptides to the secretory pathway of the recombinant expression system (e.g., a yeast recombinant expression system).

“Agent” refers to one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryote organisms, or eukaryote organisms, and agents produced therefrom.

“Agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation.

“Agroinfection” means a plant transformation method where DNA is introduced into a plant cell by using Agrobacteria A. tumefaciens or A. rhizogenes.

“BAAS” means barley alpha-amylase signal peptide, and is an example of an ERSP. One example of a BAAS is a BAAS having the amino acid sequence of SEQ ID NO:60 (NCBI Accession No. AAA32925.1).

“Biomass” refers to any measured plant product.

“Binary vector” or “binary expression vector” means an expression vector which can replicate itself in both E. coli strains and Agrobacterium strains. Also, the vector contains a region of DNA (often referred to as t-DNA) bracketed by left and right border sequences that is recognized by virulence genes to be copied and delivered into a plant cell by Agrobacterium.

“bp” or “base pair” refers to a molecule comprising two chemical bases bonded to one another forming a. For example, a DNA molecule consists of two winding strands, wherein each strand has a backbone made of an alternating deoxyribose and phosphate groups. Attached to each deoxyribose is one of four bases, i.e., adenine (A), cytosine (C), guanine (G), or thymine (T), wherein adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.

“C-terminal” refers to the free carboxyl group (i.e., —COOH) that is positioned on the terminal end of a polypeptide.

“C_E” refers to a peptide subunit having an N-terminus that is operably linked to the sixth cysteine residue that participates in the disulfide bond formation the cystine knot motif (i.e., C^VI), in the CK architecture according to Formula (II).

As used herein, the letter “C” with a superscript roman numeral, i.e., “C^I”, “C^II”, “C^III”, “C^IV”, “C^V”, and “C^VI”, refers to the cysteine residues that take part in disulfide bond formation, wherein cysteine residues C^Iand C^IVare connected by a first disulfide bond; C^IIand C^Vare connected by a second disulfide bond; and C^IIIand C^VIare connected by a third disulfide bond; wherein the first disulfide bond, the second disulfide bond, and the third disulfide bond have a disulfide bond topology that forms a cystine knot motif, and wherein the first disulfide bond, second disulfide bond, and third disulfide bond are the only disulfide bonds that form the cystine knot motif. Accordingly, a modifiable CRP can have one or more cysteine residues that are operable to form one or more non-CK disulfide bonds, wherein the one or more non-CK disulfide bonds are not the first disulfide bond, the second disulfide bond, or the third disulfide bond, and wherein the one or more non-CK disulfide bonds do not form the CK motif Thus, the superscript roman numerals I, II, III, IV, V, and VI indicate a given cysteine residue that is the first, second, third, fourth, fifth, and sixth cysteine residue to take part in disulfide bond formation, respectively, and wherein those disulfide bonds are the aforementioned first disulfide bond, second disulfide bond, and third disulfide bond form a cystine knot motif. The cysteine residues labeled as “C^III”, “C^II”, “C^III”, “C^IV”, “C^V”, and “C^VI”, and/or the superscript roman numerals I, II, III, IV, V, and VI are not meant to indicate, nor should they be construed as the first, second, third, fourth, fifth, and sixth cysteine residues in an amino acid sequence, as other cysteine residues may be present in a modifiable CRP, regardless of whether those other cysteine residues form a non-CK disulfide bond. For example, a modifiable CRP may have one or more cysteine residues present in its amino acid sequence (reading from the N-terminus to the C-terminus) that occur in the amino acid sequence before the C^Iresidue. Likewise, one or more cysteine residues may be present in the peptide subunits, that may or may not form a non-CK disulfide bond.

“cDNA” or “copy DNA” or “complementary DNA” refers to a molecule that is complementary to a molecule of RNA. In some embodiments, cDNA may be either single-stranded or double-stranded. In some embodiments, cDNA can be a double-stranded DNA synthesized from a single stranded RNA template in a reaction catalyzed by a reverse transcriptase. In yet other embodiments, “cDNA” refers to all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3′ and 5′ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the protein. In some embodiments, “cDNA” refers to a DNA that is complementary to and derived from an mRNA template.

“CEW” refers to Corn earworm.

“CK architecture” or “cystine knot architecture” refers to the shared structural similarity between peptides, polypeptides, or proteins having an CK motif, e.g., comprising three disulfide bonds, and wherein cysteines C^Iand C^IV; C^IIand C^V; and C^IIIand C^VIare connected by a disulfide bond. In some embodiments, “shared structural similarity” refers to the presence of shared structural features, e.g., the presence and/or identity of particular amino acids at particular positions. In yet other embodiments the term “shared structural similarity” refers to presence and/or identity of structural elements (for example: loops, sheets, helices, H-bond donors, H-bond acceptors, glycosylation patterns, salt bridges, and disulfide bonds). In some embodiments, the term “shared structural similarity” refers to three dimensional arrangement and/or orientation of atoms or moieties relative to one another (for example: distance and/or angles between or among them between an agent of interest and a reference agent). In some embodiments, the CK architecture comprises the following scaffold, framework, architecture, and/or backbone: N_E—C^I-L₁-C^II-L₂-C^III-L3-C^IV-L4-C^V-L₅-C^VI-C_E; wherein C^Ito C^VIare cysteine residues; wherein cysteine residues C^Iand C^IVare connected by a first disulfide bond; C^IIand C^Vare connected by a second disulfide bond; and C^IIIand C^VIare connected by a third disulfide bond; wherein the first disulfide bond, the second disulfide bond, and the third disulfide bond have a disulfide bond topology that forms a cystine knot motif, wherein the first disulfide bond, second disulfide bond, and third disulfide bond are the only disulfide bonds that form the cystine knot motif, wherein N_E, L₁, L₂, L₃, L₄, L₅, and C_Eare peptide subunits comprising an amino acid sequence having a length of 1 to 13 amino acid residues; and wherein N_E, L₃, C_E, or any combination thereof, are optionally absent.

“Cleavable Linker” see Linker.

“Cloning” refers to the process and/or methods concerning the insertion of a DNA segment (e.g., usually a gene of interest, for example dvp) from one source and recombining it with a DNA segment from another source (e.g., usually a vector, for example, a plasmid) and directing the recombined DNA, or “recombinant DNA” to replicate, usually by transforming the recombined DNA into a bacteria or yeast host.

“Coding sequence” or “CDS” refers to a polynucleotide or nucleic acid sequence that can be transcribed (e.g., in the case of DNA) or translated (e.g., in the case of mRNA) into a peptide, polypeptide, or protein, when placed under the control of appropriate regulatory sequences and in the presence of the necessary transcriptional and/or translational molecular factors. The boundaries of the coding sequence are determined by a translation start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A transcription termination sequence will usually be located 3′ to the coding sequence. In some embodiments, a coding sequence may be flanked on the 5′ and/or 3′ ends by untranslated regions. In some embodiments, a coding sequence can be used to produce a peptide, a polypeptide, or a protein product. In some embodiments, the coding sequence may or may not be fused to another coding sequence or localization signal, such as a nuclear localization signal. In some embodiments, the coding sequence may be cloned into a vector or expression construct, may be integrated into a genome, or may be present as a DNA fragment.

“Codon optimization” refers to the production of a gene in which one or more endogenous, native, and/or wild-type codons are replaced with codons that ultimately still code for the same amino acid, but that are of preference in the corresponding host.

“Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides as understood by those of skill in the art. Thus, two sequences are “complementary” to one another if they are capable of hybridizing to one another to form a stable anti-parallel, double-stranded nucleic acid structure. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions. Thus, the polynucleotide whose sequence 5′-TATAC-3′ is complementary to a polynucleotide whose sequence is 5′-GTATA-3′.

“Conditioned medium” means the cell culture medium which has been used by cells and is enriched with cell derived materials but does not contain cells.

“Copy number” refers to the number of identical copies of a vector, an expression cassette, an amplification unit, a gene or indeed any defined nucleotide sequence, that are present in a host cell at any time. For example, in some embodiments, a gene or another defined chromosomal nucleotide sequence may be present in one, two, or more copies on the chromosome. An autonomously replicating vector may be present in one, or several hundred copies per host cell.

“Culture” or “cell culture” refers to the maintenance of cells in an artificial, in vitro environment.

“Culturing” refers to the propagation of organisms on or in various kinds of media. For example, the term “culturing” can mean growing a population of cells under suitable conditions in a liquid or solid medium. In some embodiments, culturing refers to fermentative recombinant production of a heterologous polypeptide of interest and/or other desired end products (typically in a vessel or reactor).

“Cystine” refers to an oxidized cysteine-dimer. Cystines are sulfur-containing amino acids obtained via the oxidation of two cysteine molecules, and are linked with a disulfide bond.

“Cystine knot motif” or “CK motif” refers to protein structural motif comprising 3 disulfide bonds. The term “cystine-knot motif” as used herein refers to a structural motif containing 3 disulfide bonds: a first disulfide bond, a second disulfide bond, and a third disulfide bond wherein the sections of peptide that occur between two of the disulfide bonds form a loop, through which a third disulfide bond passes, forming a rotaxane substructure. The first disulfide bond occurs between cysteine residues C^Iand C^IV; the second disulfide bond occurs between cysteine residues C^IIand C^V; and the third disulfide bond occurs between cysteine residues C^IIIand C^VI; wherein the first disulfide bond, second disulfide bond, and third disulfide bond have a disulfide bond topology that forms the cystine knot motif, and wherein the first disulfide bond, the second disulfide bond, and the third disulfide bond are the only disulfide bonds that form the cystine knot motif. In some embodiments, the disulfide bond topology forms one of the following cystine knot motifs: an inhibitor cystine knot (ICK) motif, a growth factor cystine knot (GFCK) motif, or a cyclic cystine knot (CCK) motif.

“Dc1a” or “Mu-diguetoxin-Dc1a” refers to a polypeptide isolated from the American Desert Spider (Diguetia canities), also known as “the desert bush spider.” One example of a wild-type Mu-diguetoxin-Dc1a is a polypeptide having the amino acid sequence of SEQ ID NO:1 (NCBI Accession No. P49126.1).

“Defined medium” means a medium that is composed of known chemical components but does not contain crude proteinaceous extracts or by-products such as yeast extract or peptone.

“Degeneracy” or “codon degeneracy” refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies. As a result of the degeneracy of the genetic code, many nucleic acid sequences can encode a given polypeptide with a particular activity; such functionally equivalent variants are contemplated herein.

“Disulfide bond” or “disulfide bridges” refers to a covalent bond between two cysteine amino acids derived by the coupling of two thiol groups on their side chains. In some embodiments, a disulfide bond occurs via the oxidative folding of two different thiol groups (—SH) present in a polypeptide, e.g., a CRIP. In some embodiments, a polypeptide can comprise at least six different thiol groups (i.e., six cysteine residues each containing a thiol group); thus, in some embodiments, a polypeptide can form three, or more intramolecular disulfide bonds.

“Disulfide bond topology” or “disulfide bond linkage pattern” or “disulfide bond connectivity” refers to the linking pattern of disulfide bonds and cysteine residues. In some embodiments, a CRIP with the CK architecture of Formula (II) comprises six conserved cysteine residues (numbered I-VI) that form three disulfide bonds with the following disulfide bond connectivities: C^Iand C^IV; C^IIand C^V; and C^IIIand C^VI. In some embodiments, the disulfide bonding connectivity is topologically constant, meaning the disulfide bonds can only be changed by unlinking one or more disulfides such as using redox conditions.

“Double expression cassette” refers to two DVP expression cassette s contained on the same vector.

“Double transgene peptide expression vector” or “double transgene expression vector” means a yeast expression vector that contains two copies of the DVP expression cassette.

“DNA” refers to deoxyribonucleic acid, comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form. For example, one or more nucleotides creates a polynucleotide.

“dNTPs” refers to the nucleoside triphosphates that compose DNA and RNA.

“dvp” or “Mu-diguetoxin-Dc1a variant polynucleotide” or “Dc1a variant polynucleotide” or “variant Mu-diguetoxin-Dc1a polynucleotide” refers to a polynucleotide sequence operable to encodes a DVP. The term “Mu-diguetoxin-Dc1a variant polynucleotide” when used to describe the Mu-diguetoxin-Dc1a variant polynucleotide sequence contained in a DVP ORF, its inclusion in a vector, and/or when describing the polynucleotides encoding an insecticidal protein, is described as “dvp” and/or “Dvp”.

“DVP” or “Mu-diguetoxin-Dc1a Variant Polypeptides” refer to peptide, polypeptide, or protein mutants or variants that differ in some way from the wild-type mature Mu-diguetoxin-Dc1a (SEQ ID NO:2); for example, in some embodiments, this variance can be an amino acid substitution, amino acid deletion/insertion, and/or a mutation or variance to a polynucleotide operable to encode the wild-type Mu-diguetoxin-Dc1a. The result of this variation is a non-naturally occurring polypeptide and/or polynucleotide sequence encoding the same that possesses insecticidal activity against one or more insect species, relative to the wild-type Mu-diguetoxin-Dc1a.

“DVP expression cassette” refers to one or more regulatory elements such as promoters; enhancer elements; mRNA stabilizing polyadenylation signal; an internal ribosome entry site (IRES); introns; post-transcriptional regulatory elements; and a polynucleotide operable to encode a DVP, e.g., a DVP ORF. For example, one example of a DVP expression cassette is one or more segments of DNA that contains a polynucleotide segment operable to express a DVP, a ADH1 promoter, a LAC4 terminator, and an alpha-MF secretory signal. A DVP expression cassette contains all of the nucleic acids necessary to encode a DVP or a DVP-insecticidal protein.

“DVP ORF” refers to a polynucleotide operable to encode a DVP, or a DVP-insecticidal protein.

“DVP ORF diagram” refers to the composition of one or more DVP ORFs, as written out in diagram or equation form. For example, a “DVP ORF diagram” can be written out as using acronyms or short-hand references to the DNA segments contained within the expression ORF. Accordingly, in one example, a “DVP ORF diagram” may describe the polynucleotide segments encoding the ERSP, LINKER, STA, and DVP, by diagramming in equation form the DNA segments as “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide); “linker” or “L” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and “dvp” (i.e., the polynucleotide sequence encoding a DVP), respectively. An example of a DVP ORF diagram is “ersp-sta-(linker_i-dvp_j)_N,” or “ersp-(dvp_j-linker_i)_N-sta” and/or any combination of the DNA segments thereof.

“DVP-insecticidal protein” refers to any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, consisting of: (1) at least one DVP, or two or more DVPs (wherein said two or more DVPs may be the same or different); and (2) additional non-toxin peptides, polypeptides, or proteins, wherein said additional non-toxin peptides, polypeptides, or proteins e.g., in some embodiments, have the ability to do one or more of the following: increase the mortality and/or inhibit the growth of insects when the insects are exposed to a DVP-insecticidal protein, relative to a DVP alone; increase the expression of said DVP-insecticidal protein, e.g., in a host cell or an expression system; and/or affect the post-translational processing of the DVP-insecticidal protein (e.g., allow for secreted expression of the DVP-insecticidal protein). In some embodiments, a DVP-insecticidal protein can be a polymer comprising two or more DVPs. In some embodiments, a DVP-insecticidal protein can be a polymer comprising two or more DVPs, wherein the DVPs are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker. In some embodiments, a DVP-insecticidal protein can refer to a one or more DVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker (L); and/or any other combination thereof. In some embodiments, a DVP-insecticidal protein can be a non-naturally occurring protein comprising (1) a wild-type Dc1a protein; and (2) additional non-toxin peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker. In some embodiments, the DVP-insecticidal protein can comprise: (1) a DVP; and (2) an alpha mating factor peptide. For example in some embodiments, a DVP-insecticidal protein can comprise: (1) a DVP; and (2) an alpha mating factor (alpha-MF) or α-mating factor (α-MF) secretion domain (for secreted expression). In some embodiments, a DVP-insecticidal protein can comprise: (1) a DVP; and (2) a K. lactis α-mating factor (α-MF) secretion domain (for secreted expression). In some embodiments, a DVP-insecticidal protein can comprise: (1) two or more DVPs, wherein the DVPs are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker; and wherein the DVPs are the same or different; and (2) an alpha-MF, e.g., a K. lactis α-mating factor (α-MF) secretion domain (for secreted expression).

“DVP construct” refers to the three-dimensional arrangement/orientation of peptides, polypeptides, and/or motifs of operably linked polypeptide segments (e.g., a DVP-insecticidal protein). For example, a DVP ORF can include one or more of the following components or motifs: a DVP; an endoplasmic reticulum signal peptide (ERSP); a linker peptide (L); a translational stabilizing protein (STA); or any combination thereof. And, as used herein, the term “DVP construct” is used to describe the designation and/or orientation of the structural motif. In other words, the DVP construct describes the arrangement and orientation of the components or motifs contained within a given DVP ORF. For example, in some embodiments, a DVP construct describes, without limitation, the orientation of one of the following DVP-insecticidal proteins: ERSP-DVP; ERSP-(DVP)_N; ERSP-DVP-L; ERSP-(DVP)_N-L; ERSP-(DVP-L)_N; ERSP-L-DVP; ERSP-L-(DVP)_N; ERSP-(L-DVP)_N; ERSP-STA-DVP; ERSP-STA-(DVP)_N; ERSP-DVP-STA; ERSP-(DVP)_N—STA; ERSP-(STA-DVP)_N; ERSP-(DVP-STA)_N; ERSP-L-DVP-STA; ERSP-L-STA-DVP; ERSP-L-(DVP-STA)_N; ERSP-L-(STA-DVP)_N; ERSP-L-(DVP)_N—STA; ERSP-(L-DVP)_N—STA; ERSP-(L-STA-DVP)_N; ERSP-(L-DVP-STA)_N; ERSP-(L-STA)_N-DVP; ERSP-(L-DVP)_N—STA; ERSP-STA-L-DVP; ERSP-STA-DVP-L; ERSP-STA-L-(DVP)_N; ERSP-(STA-L)_N-DVP; ERSP-STA-(L-DVP)_N; ERSP-(STA-L-DVP)_N; ERSP-STA-(DVP)_N-L; ERSP-STA-(DVP-L)_N; ERSP-(STA-DVP)_N-L; ERSP-(STA-DVP-L)_N; ERSP-DVP-L-STA; ERSP-DVP-STA-L; ERSP-(DVP)_N—STA-L ERSP-(DVP-L)_N-STA; ERSP-(DVP-STA)_N-L; ERSP-(DVP-L-STA)_N; or ERSP-(DVP-STA-L)_N; wherein N is an integer ranging from 1 to 200.

“ELISA” or “iELISA” means an assay protocol in which the samples are fixed to the surface of a plate and then detected as follows: a primary antibody is applied followed by a secondary antibody conjugated to an enzyme which converts a colorless substrate to colored substrate which can be detected and quantified across samples. During the protocol, antibodies are washed away such that only those that bind to their epitopes remain for detection. The samples, in our hands, are predominantly proteins, and ELISA allows for the quantification of the amount of protein recovered.

“Endogenous” refers to a polynucleotide, peptide, polypeptide, protein, or process that naturally occurs and/or exists in an organism, e.g., a molecule or activity that is already present in the host cell before a particular genetic manipulation.

“Enhancer element” refers to a DNA sequence operably linked to a promoter, which can exert increased transcription activity on the promoter relative to the transcription activity that results from the promoter in the absence of the enhancer element.

“ER” or “Endoplasmic reticulum” is a subcellular organelle common to all eukaryotes where some post translation modification processes occur.

“ERSP” or “Endoplasmic reticulum signal peptide” is an N-terminus sequence of amino acids that-during protein translation of the mRNA molecule encoding a DVP—is recognized and bound by a host cell signal-recognition particle, which moves the protein translation ribosome/mRNA complex to the ER in the cytoplasm. The result is the protein translation is paused until it docks with the ER where it continues and the resulting protein is injected into the ER.

“ersp” refers to a polynucleotide encoding the peptide, ERSP.

“ER trafficking” means transportation of a cell expressed protein into ER for post-translational modification, sorting and transportation.

“Expression cassette” refers to all the DNA elements necessary to complete transcription of a transgene or a heterologous polynucleotide—e.g., a polynucleotide operable to encode a DVP in a recombinant expression system. Thus, in some embodiments, an “expression cassette” refers to a (1) a DNA sequence of interest, e.g., a heterologous polynucleotide operable to encode a DVP; and one or more of the following: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements. The combination (1) with at least one of (2)-(6) is called an “expression cassette.”

For example, in some embodiments, an expression cassette can be (1) a heterologous polynucleotide operable to encode a DVP; and further comprising one or more: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements.

In some embodiments, an expression cassette can be (1) one or more heterologous polynucleotides operable to encode a DVP; and further comprising one or more: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements; wherein each of the one or more heterologous polynucleotides operable to encode a DVP, further comprises one or more of (2)-(6); wherein the DVP can be the same or different.

For example, in some embodiments, an expression cassette can refer to (1) a first heterologous polynucleotide operable to encode a DVP, and one or more additional heterologous polynucleotide operable to encode a DVP; further comprising one or more of: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements; wherein either the first heterologous polynucleotide operable to encode a DVP, and the one or more additional heterologous polynucleotide operable to encode a DVP further comprises one or more of (2)-(6); or wherein each of the first heterologous polynucleotide operable to encode a DVP, and each of the one or more additional heterologous polynucleotide operable to encode a DVP, each individually further comprises one or more of (2)-(6); wherein the DVP can be the same or different.

In alternative embodiments, there are two expression cassettes, each expression cassette comprising a heterologous polynucleotide operable to encode a DVP (i.e., a double expression cassette), wherein the DVP can be the same or different.

In other embodiments, there are three expression cassettes, each expression cassette comprising a heterologous polynucleotide operable to encode a DVP (i.e., a triple expression cassette); wherein the DVP can be the same or different.

In some embodiments, a double expression cassette can be generated by subcloning a second expression cassette into a vector containing a first expression cassette. In some embodiments, a triple expression cassette can be generated by subcloning a third expression cassette into a vector containing a first and a second expression cassette. Methods concerning expression cassettes and cloning techniques are well-known in the art and described herein.

“FECT” means a transient plant expression system using Foxtail mosaic virus with elimination of coating protein gene and triple gene block.

“GFP” means a green fluorescent protein from the jellyfish, Aequorea victoria.

“Growth medium” refers to a nutrient medium used for growing cells in vitro.

“Gut” as used herein can refer to any organ, structure, tissue, cell, extracellular matrix, and/or space comprising the gut, for example: the foregut, e.g., mouth, pharynx, esophagus, crop, proventriculus, or crop; the midgut, e.g., midgut caecum, ventriculus; the hindgut, e.g., pylorum, ileum, rectum or anus; the peritrophic membrane; microvilli; the basement membrane; the muscle layer; Malpighian tubules; or rectal ampulla.

“Homologous” refers to Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ×100. Homologous refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology.

The term “homology,” when used in relation to nucleic acids, refers to a degree of complementarity. There may be partial homology, or complete homology and thus identical. “Sequence identity” refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences.

“ICK motif” or “ICK motif protein” refers to a 16 to 60 amino acid peptide with at least 6 half-cystine core amino acids having three disulfide bridges. In some embodiments, the three disulfide bridges are covalent bonds and of the six half-cystine residues the covalent disulfide bonds are between the first and fourth, the second and fifth, and the third and sixth half-cystines, of the six core half-cystine amino acids starting from the N-terminal amino acid. In some embodiments, peptides possessing this motif comprise a beta-hairpin secondary structure, normally composed of residues situated between the fourth and sixth core half-cystines of the motif, wherein the hairpin is stabilized by the structural crosslinking provided by the motif's three disulfide bonds. In some embodiments, additional cysteine/cystine or half-cystine amino acids may be present within the inhibitor cystine knot motif.

“Identity” refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing said sequences. The term “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by any one of the myriad methods known to those having ordinary skill in the art, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the disclosures of which are incorporated herein by reference in their entireties. Furthermore, methods to determine identity and similarity are codified in publicly available computer programs. For example in some embodiments, methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990), the disclosures of which are incorporated herein by reference in their entireties.

“in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

“Inactive” refers to a condition wherein something is not in a state of use, e.g., lying dormant and/or not working. For example, when used in the context of a gene or when referring to a gene, the term inactive means said gene is no longer actively synthesizing a gene product, having said gene product translated into a protein, or otherwise having the gene perform its normal function. For example, in some embodiments, the term inactive can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and/or inhibition and/or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.

“Increasing” or “increase” or “increased” or “increases” refers to making something (e.g., the expression of peptide, polypeptide, or protein) greater in size, amount, intensity, or degree. For example, in some embodiments, the removal of one or more disulfide bonds from a modifiable CRP not having a CK architecture according to Formula (II), can result in the creation of a recombinant CRP having a CK architecture according to Formula (II), wherein having a CK architecture according to Formula (II) results in the following effect: an increase in the level of expression of the recombinant CRP, and/or an increase in the yield of the recombinant CRP, relative to the modifiable CRP not having the CK architecture according to Formula (II).

Thus, in some embodiments, the terms “increased level of expression” or “an increase in the level of expression” or “increased yield” or “an increase in yield,” in a recombinant CRP having the CK architecture according to Formula (II), refers to an increase that is at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 20.25%, at least about 2.5%, at least about 20.75%, at least about 3%, at least about 30.25%, at least about 3.5%, at least about 3.75%, at least about 4%, at least about 4 0.25%, at least about 4.5%, at least about 4.75%, at least about 5%, at least about 5.2 5%, at least about 5.5%, at least about 5.75%, at least about 6%, at least about 6 0.25%, at least about 6.5%, at least about 6.75%, at least about 7%, at least about 7.25%, at least about 7.5%, at least about 7.75%, at least about 8%, at least about 8.25%, at least about 8.5%, at least about 8.75%, at least about 9%, at least about 9 0.25%, at least about 9.5%, at least about 9 0.75%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or a greater than a 100%, in the amount of protein, the level of expression of protein, and/or the yield of protein in the recombinant CRP having the CK architecture according to Formula (II), relative to the amount of protein, the level of expression of protein, and/or the yield of protein in the modifiable CRP that does not have the CK architecture according to Formula (II).

“Inoperable” refers to the condition of a thing not functioning, malfunctioning, or no longer able to function. For example, when used in the context of a gene or when referring to a gene, the term inoperable means said gene is no longer able to operate as it normally would, either permanently or transiently. For example, “inoperable,” in some embodiments, means that a gene is no longer able to synthesize a gene product, having said gene product translated into a protein, or is otherwise unable to gene perform its normal function. For example, in some embodiments, the term inoperable can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and/or inhibition and/or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.

“Insect” includes all organisms in the class “Insecta.” The term “pre-adult” insects refers to any form of an organism prior to the adult stage, including, for example, eggs, larvae, and nymphs. As used herein, the term “insect refers to any arthropod and nematode, including acarids, and insects known to infest all crops, vegetables, and trees and includes insects that are considered pests in the fields of forestry, horticulture and agriculture. Examples of specific crops that might be protected with the methods disclosed herein are soybean, corn, cotton, alfalfa and the vegetable crops. A list of specific crops and insects is enclosed herein.

“Insect gut environment” or “gut environment” means the specific pH and proteinase conditions found within the fore, mid or hind gut of an insect or insect larva.

“Insect hemolymph environment” means the specific pH and proteinase conditions of found within an insect or insect larva.

As used herein, the term “insecticidal” is generally used to refer to the ability of a polypeptide or protein used herein, to increase mortality or inhibit growth rate of insects. As used herein, the term “nematicidal” refers to the ability of a polypeptide or protein used herein, to increase mortality or inhibit the growth rate of nematodes. In general, the term “nematode” comprises eggs, larvae, juvenile and mature forms of said organism.

“Insecticidal activity” means that upon or after exposing the insect to compounds, agents, or peptides, the insect either dies stops or slows its movement; stops or slows its feeding; stops or slows its growth; becomes confused (e.g., with regard to navigation, locating food, sleeping behaviors, and/or mating); fails to pupate; interferes with reproduction; and/or precludes the insect from producing offspring and/or precluding the insect from producing fertile offspring.

“Integrative expression vector” or “integrative vector” means a yeast expression vector which can insert itself into a specific locus of the yeast cell genome and stably becomes a part of the yeast genome.

“Intervening linker” refers to a short peptide sequence in the protein separating different parts of the protein, or a short DNA sequence that is placed in the reading frame in the ORF to separate the upstream and downstream DNA sequences. For example, in some embodiments, an intervening linker may be used allowing proteins to achieve their independent secondary and tertiary structure formation during translation. In some embodiments, the intervening linker can be either resistant or susceptible to cleavage in plant cellular environments, in the insect and/or lepidopteran gut environment, and in the insect hemolymph and lepidopteran hemolymph environment.

“Isolated” refers to separating a thing and/or a component from its natural environment, e.g., a toxin isolated from a given genus or species means that toxin is separated from its natural environment.

“Kappa-ACTX peptide” or “x-ACTX” (all used interchangeably) refers to a peptide belonging to a family of insecticidal inhibitor cystine knot (ICK) peptides that have been isolated from Australian funnel-web spiders belonging to the Atracinae subfamily. One such spider is the Australian Blue Mountains Funnel-web Spider, which has the scientific name Haydronyche versuta. An exemplary wild-type Kappa-ACTX peptide is provided herein, having the amino acid sequence: “AICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP” (SEQ ID NO: 198) (UniProtKB/Swiss-Prot No. P82228.1).

“kb” refers to kilobase, i.e., 1000 bases. As used herein, the term “kb” means a length of nucleic acid molecules. For example, 1 kb refers to a nucleic acid molecule that is 1000 nucleotides long. A length of double-stranded DNA that is 1 kb long, contains two thousand nucleotides (i.e., one thousand on each strand). Alternatively, a length of single-stranded RNA that is 1 kb long, contains one thousand nucleotides.

“kDa” refers to kilodalton, a unit equaling 1,000 daltons; a “Dalton” or “dalton” is a unit of molecular weight (MW).

“Knock in” or “knock-in” or “knocks-in” or “knocking-in” refers to the replacement of an endogenous gene with an exogenous or heterologous gene, or part thereof. For example, in some embodiments, the term “knock-in” refers to the introduction of a nucleic acid sequence encoding a desired protein to a target gene locus by homologous recombination, thereby causing the expression of the desired protein. In some embodiments, a “knock-in” mutation can modify a gene sequence to create a loss-of-function or gain-of-function mutation. The term “knock-in” can refer to the procedure by which a exogenous or heterologous polynucleotide sequence or fragment thereof is introduced into the genome, (e.g., “they performed a knock-in” or “they knocked-in the heterologous gene”), or the resulting cell and/or organism (e.g., “the cell is a “knock-in” or “the animal is a “knock-in”).

“Knock out” or “knockout” or “knock-out” or “knocks-out” or “knocking-out” refers to a partial or complete suppression of the expression gene product (e.g., mRNA) of a protein encoded by an endogenous DNA sequence in a cell. In some embodiments, the “knock-out” can be effectuated by targeted deletion of a whole gene, or part of a gene encoding a peptide, polypeptide, or protein. As a result, the deletion may render a gene inactive, partially inactive, inoperable, partly inoperable, or otherwise reduce the expression of the gene or its products in any cell in the whole organism and/or cell in which it is normally expressed. The term “knock-out” can refer to the procedure by which an endogenous gene is made completely or partially inactive or inoperable (e.g., “they performed a knock-out” or “they knocked-out the endogenous gene”), or the resulting cell and/or organism (e.g., “the cell is a “knock-out” or “the animal is a “knock-out”).

“Knockdown dose 50” or “KD₅₀” refers to the median dose required to cause paralysis or cessation of movement in 50% of a population, for example a population of Musca domestica (common housefly) and/or Aedes aegypti (mosquito).

“I” or “linker” refers to a nucleotide encoding intervening linker peptide.

“L₁” refers to a peptide subunit located between the first cysteine and second cysteine residues that participate in the disulfide bond formation the cystine knot motif (i.e., C^Iand C^II) in the CK architecture according to Formula (II).

“L₂” refers to a peptide subunit located between the second cysteine and third cysteine residues that participate in the disulfide bond formation the cystine knot motif (i.e., C^Iand C^III) in the CK architecture according to Formula (II).

“L₃” refers to a peptide subunit located between the third cysteine and fourth cysteine residues that participate in the disulfide bond formation the cystine knot motif (i.e., C^IIIand C^IV) in the CK architecture according to Formula (II).

“L₄” refers to a peptide subunit located between the fourth cysteine and fifth cysteine residues that participate in the disulfide bond formation the cystine knot motif (i.e., C^IVand C^V) in the CK architecture according to Formula (II).

“L₅” refers to a peptide subunit located between the fifth cysteine and sixth cysteine residues that participate in the disulfide bond formation the cystine knot motif (i.e., C^Vand C^VI) in the CK architecture according to Formula (II).

“L” in the proper context refers to an intervening linker peptide, which links a translational stabilizing protein (STA) with an additional polypeptide, e.g., a DVP, and/or multiple DVPs. When referring to amino acids, “L” can also mean leucine.

“LAC4 promoter” or “Lac4 promoter” or “pLac4” refers to a DNA segment comprised of the promoter sequence derived from the K. lactis β-galactosidase gene. The LAC4 promoters is strong and inducible reporter that is used to drive expression of exogenous genes transformed into yeast.

“LAC4 terminator” or “Lac4 terminator” refers to a DNA segment comprised of the transcriptional terminator sequence derived from the K. lactis β-galactosidase gene.

“Lepidopteran gut environment” means the specific pH and proteinase conditions of found within the fore, mid or hind gut of a lepidopteran insect or larva.

“Lepidopteran hemolymph environment” means the specific pH and proteinase conditions of found within lepidopteran insect or larva.

“LD₂₀” refers to a dose required to kill 20% of a population.

“LD₅₀” refers to lethal dose 50 which means the dose required to kill 50% of a population.

“Linker” or “LINKER” or “peptide linker” or “L” or “intervening linker” refers to a short peptide sequence operable to link two peptides together. Linker can also refer to a short DNA sequence that is placed in the reading frame of an ORF to separate an upstream and downstream DNA sequences. In some embodiments, a linker can be cleavable by an insect protease. In some embodiments, a linker may allow proteins to achieve their independent secondary and tertiary structure formation during translation. In some embodiments, the linker can be either resistant or susceptible to cleavage in plant cellular environments, in the insect and/or lepidopteran gut environment, and/or in the insect hemolymph and lepidopteran hemolymph environment. In some embodiments, a linker can be cleaved by a protease, e.g., in some embodiments, a linker can be cleaved by a plant protease (e.g., papain, bromelain, ficin, actinidin, zingibain, and/or cardosins), an insect protease, a fungal protease, a vertebrate protease, an invertebrate protease, a bacteria protease, a mammal protease, a reptile protease, or an avian protease. In some embodiments, a linker can be cleavable or non-cleavable. In some embodiments, a linker comprises a binary or tertiary region, wherein each region is cleavable by at least two types of proteases: one of which is an insect and/or nematode protease and the other one of which is a human protease. In some embodiments, a linker can have one of (at least) three roles: to cleave in the insect gut environment, to cleave in the plant cell, or to be designed not to intentionally cleave.

“Medium” (plural “media”) refers to a nutritive solution for culturing cells in cell culture.

“MOA” refers to mechanism of action.

“Modifiable CRP” refers to a cysteine rich protein having one or more non-CK disulfide bonds, in addition to a first disulfide bond, a second disulfide bond, and a third disulfide bond having a disulfide bond topology that forms a cystine knot motif, wherein the one or more non-CK disulfide bonds are not the first disulfide bond, the second disulfide bond, or the third disulfide bond, and wherein the one or more non-CK disulfide bonds do not form the CK motif. Examples of a modifiable CRP include an ApsIII protein having the amino acid sequence of a “CNSKGTPCTNADECCGGKCAYNVWNCIGGGCSKTCGY” (SEQ ID NO: 193; NCBI Accession No. P49268.1); a wild-type Kappa-ACTX peptide having the amino acid sequence: “AICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP” (SEQ ID NO: 198; UniProtKB/Swiss-Prot No. P82228.1); and or any one of SEQ ID NOs: 1-2, or 195.

“Molecular weight (MW)” refers to the mass or weight of a molecule, and is typically measured in “daltons (Da)” or kilodaltons (kDa). In some embodiments, MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical ultracentrifugation, or light scattering. In some embodiments, the SDS-PAGE method is as follows: the sample of interest is separated on a gel with a set of molecular weight standards. The sample is run, and the gel is then processed with a desired stain, followed by destaining for about 2 to 14 hours. The next step is to determine the relative migration distance (Rf) of the standards and protein of interest. The migration distance can be determined using the following equation:

$\begin{matrix} Rf = \frac{Migration distance of the protein}{Migration distance of the dye front} & Formula (III) \end{matrix}$

Next, the logarithm of the MW can be determined based on the values obtained for the bands in the standard; e.g., in some embodiments, the logarithm of the molecular weight of an SDS-denatured polypeptide and its relative migration distance (Rf) is plotted into a graph. After plotting the graph, interpolating the value derived will provide the molecular weight of the unknown protein band.

“Motif” refers to a polynucleotide or polypeptide sequence that is implicated in having some biological significance and/or exerts some effect or is involved in some biological process.

“Multiple cloning site” or “MCS” refers to a segment of DNA found on a vector that contains numerous restriction sites in which a DNA sequence of interest can be inserted.

“Mutant” refers to an organism, DNA sequence, peptide sequence, or polypeptide sequence, that has an alteration (for example, in the DNA sequence), which causes said organism and/or sequence to be different from the naturally occurring or wild-type organism and/or sequence. For example, a wild-type Mu-diguetoxin-Dc1a polypeptide can be altered resulting in a non-naturally occurring DVP.

“N_E” refers to a peptide subunit having a C-terminus that is operably linked to the first cysteine residue that participates in the disulfide bond formation the cystine knot motif (i.e., C^I), in the CK architecture according to Formula (II).

“N-terminal” refers to the free amine group (i.e., —NH₂) that is positioned on beginning or start of a polypeptide.

“NCBI” refers to the National Center for Biotechnology Information.

“nm” refers to nanometers.

“Non-Polar amino acid” is an amino acid that is weakly hydrophobic and includes glycine, alanine, proline, valine, leucine, isoleucine, phenylalanine and methionine. Glycine or gly is the most preferred non-polar amino acid for the dipeptides of this invention.

“Normalized peptide yield” means the peptide yield in the conditioned medium divided by the corresponding cell density at the point the peptide yield is measured. The peptide yield can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg/L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu·sec. The cell density can be represented by visible light absorbance of the culture at wavelength of 600 nm (OD600).

“OD” refers to optical density. Typically, OD is measured using a spectrophotometer.

“OD660 nm” or “OD_660nm” refers to optical densities at 660 nanometers (nm).

“One letter code” means the peptide sequence which is listed in its one letter code to distinguish the various amino acids in the primary structure of a protein: alanine=A, arginine=R, asparagine=N, aspartic acid=D, asparagine or aspartic acid=B, cysteine=C, glutamic acid=E, glutamine=Q, glutamine or glutamic acid=Z, glycine=G, histidine=H, isoleucine=I, leucine=L, lysine=K, methionine=M, phenylalanine=F, proline=P, serine=S, threonine=T, tryptophan=W, tyrosine=Y, and valine=V.

“Operable” refers to the ability to be used, the ability to do something, and/or the ability to accomplish some function or result. For example, in some embodiments, “operable” refers to the ability of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence or gene to encode a peptide, polypeptide, and/or protein. For example, in some embodiments, a polynucleotide may be operable to encode a protein, which means that the polynucleotide contains information that imbues it with the ability to create a protein (e.g., by transcribing mRNA, which is in turn translated to protein).

“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, in some embodiments, operably linked can refer to two or more DNA, peptide, or polypeptide sequences. In other embodiments, operably linked can mean that the two adjacent DNA sequences are placed together such that the transcriptional activation of one DNA sequence can act on the other DNA sequence. In yet other embodiments, the term “operably linked” can refer to two or more peptides and/or polypeptides, wherein said two or more peptides and/or polypeptides are connected in such a way as to yield a single polypeptide chain; alternatively, the term operably linked can refer to two or more peptides that are connected in such a way that one peptide exerts some effect on the other. In yet other embodiments, operably linked can refer to two adjacent DNA sequences are placed together such that the transcriptional activation of one can act on the other.

“ORF” or “open reading frame” refers to a length of RNA or DNA sequence, between a translation start signal (e.g., AUG or ATG, respectively) and any one or more of the known termination codons, which encodes one or more polypeptide sequences. Put another way, the ORF describes the frame of reference as seen from the point of view of a ribosome translating the RNA code, insofar that the ribosome is able to keep reading (i.e., adding amino acids to the nascent protein) because it has not encountered a stop codon. Thus, “open reading frame” or “ORF” refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. Here, the terms “initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides (i.e., a codon) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

In some embodiments, an ORF is a continuous stretch of codons that begins with a start codon (usually ATG for DNA, and AUG for RNA) and ends at a stop codon (usually UAA, UAG or UGA). In other embodiments, an ORF can be length of RNA or DNA sequence, between a translation start signal (e.g., AUG or ATG) and any one or more of the known termination codons, wherein said length of RNA or DNA sequence encodes one or more polypeptide sequences. In some other embodiments, an ORF can be a DNA sequence encoding a protein which begins with an ATG start codon and ends with a TGA, TAA or TAG stop codon. ORF can also mean the translated protein that the DNA encodes. Generally, those having ordinary skill in the art distinguish the terms “open reading frame” and “ORF,” from the term “coding sequence,” based upon the fact that the broadest definition of “open reading frame” simply contemplates a series of codons that does not contain a stop codon. Accordingly, while an ORF may contain introns, the coding sequence is distinguished by referring to those nucleotides (e.g., concatenated exons) that can be divided into codons that are actually translated into amino acids by the ribosomal translation machinery (i.e., a coding sequence does not contain introns); however, as used herein, the terms “coding sequence”; “CDS”; “open reading frame”; and “ORF,” are used interchangeably.

“Out-recombined” or “out-recombination” refers to the removal of a gene and/or polynucleotide sequence (e.g., an endogenous gene) that is flanked by two site-specific recombination sites (e.g., the 5′- and 3′-nucleotide sequence of a target gene that is homologous to the homology arms of a target vector) during in vivo homologous recombination. See “knockout.”

“Peptide expression vector” means a host organism expression vector which contains a heterologous peptide transgene.

“Peptide expression yeast strain”, “peptide expression strain” or “peptide production strain” means a yeast strain which can produce a heterologous peptide.

“Peptide Linker” see Linker.

“Peptide subunit” refers to an amino acid sequence upstream, downstream, and/or between one or more cysteine residues in a peptide, polypeptide, or protein. In some embodiments, a peptide subunit is upstream, downstream, and/or between cysteine residues in a recombinant CRP having a CK architecture according to Formula (II). In some embodiments, a peptide subunit can have a length of 1 to 13 amino acid residues. In yet other embodiments, a peptide subunit can have a length of 13 or more amino acid residues. In some embodiments, peptide subunits in a recombinant CRP comprising the CK architecture according to Formula (II) are designated as N_E, L₁, L₂, L₃, L₄, L₅, and C_E.

“Peptide transgene” or “insecticidal peptide transgene” or “insecticidal protein transgene” or “Mu-diguetoxin-Dc1a variant transgene” refers to a DNA sequence that encodes an DVP and can be translated in a biological expression system.

“Peptide yield” means the insecticidal peptide concentration in the conditioned medium which is produced from the cells of a peptide expression yeast strain. It can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg/L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu·sec.

“Pest” includes, but is not limited to: insects, fungi, bacteria, nematodes, mites, ticks, and the like.

“Pesticidally-effective amount” refers to an amount of the pesticide that is able to bring about death to at least one pest, or to noticeably reduce pest growth, feeding, or normal physiological development. This amount will vary depending on such factors as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop, or agricultural site to be treated, the environmental conditions, and the method, rate, concentration, stability, and quantity of application of the pesticidally-effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of pest infestation.

“Pharmaceutically acceptable salt” refers to a compound that is modified by making acid or base salts thereof.

“Plant” shall mean whole plants, plant tissues, plant cells, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

“Plant transgenic protein” means a protein from a heterologous species that is expressed in a plant after the DNA or RNA encoding it was delivered into one or more of the plant cells.

“Plant-incorporated protectant” or “PIP” means an insecticidal protein produced by transgenic plants, and the genetic material necessary for the plant to produce the protein.

“Plant cleavable linker” means a cleavable linker peptide, or a nucleotide encoding a cleavable linker peptide, which contains a plant protease recognition site and can be cleaved during the protein expression process in the plant cell.

“Plant regeneration media” means any media that contains the necessary elements and vitamins for plant growth and plant hormones necessary to promote regeneration of a cell into an embryo which can germinate and generate a plantlet derived from tissue culture. Often the media contains a selectable agent to which the transgenic cells express a selection gene that confers resistance to the agent.

“Plasmid” refers to a DNA segment that acts as a carrier for a gene of interest (e.g., dvp) and, when transformed or transfected into an organism, can replicate and express the DNA sequence contained within the plasmid independently of the host organism. Plasmids are a type of vector, and can be “cloning vectors” (i.e., simple plasmids used to clone a DNA fragment and/or select a host population carrying the plasmid via some selection indicator) or “expression plasmids” (i.e., plasmids used to produce large amounts of polynucleotides and/or polypeptides).

“Polar amino acid” is an amino acid that is polar and includes serine, threonine, cysteine, asparagine, glutamine, histidine, tryptophan and tyrosine; preferred polar amino acids are serine, threonine, cysteine, asparagine and glutamine; with serine being most highly preferred.

“Polynucleotide” refers to a polymeric-form of nucleotides (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof) of any length; e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides. As used herein, the term “polynucleotide” includes double- and single-stranded DNA, as well as double- and single-stranded RNA; it also includes modified and unmodified forms of a polynucleotide (modifications to and of a polynucleotide, for example, can include methylation, phosphorylation, and/or capping). In some embodiments, a polynucleotide can be one of the following: a gene or gene fragment (for example, a probe, primer, EST, or SAGE tag); genomic DNA; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA; ribosomal RNA; ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probe; primer or amplified copy of any of the foregoing.

In yet other embodiments, a polynucleotide can refer to a polymeric-form of nucleotides operable to encode the open reading frame of a gene.

In some embodiments, a polynucleotide can refer to cDNA.

In some embodiments, polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The structure of a polynucleotide can also be referenced to by its 5′- or 3′-end or terminus, which indicates the directionality of the polynucleotide. Adjacent nucleotides in a single-strand of polynucleotides are typically joined by a phosphodiester bond between their 3′ and 5′ carbons. However, different internucleotide linkages could also be used, such as linkages that include a methylene, phosphoramidate linkages, etc. This means that the respective 5′ and 3′ carbons can be exposed at either end of the polynucleotide, which may be called the 5′ and 3′ ends or termini. The 5′ and 3′ ends can also be called the phosphoryl (PO₄) and hydroxyl (OH) ends, respectively, because of the chemical groups attached to those ends. The term polynucleotide also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment that makes or uses a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

In some embodiments, a polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with non-natural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.

In some embodiments, a polynucleotide can also be further modified after polymerization, such as by conjugation with a labeling component. Additionally, the sequence of nucleotides in a polynucleotide can be interrupted by non-nucleotide components. One or more ends of the polynucleotide can be protected or otherwise modified to prevent that end from interacting in a particular way (e.g. forming a covalent bond) with other polynucleotides.

In some embodiments, a polynucleotide can be composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T). Uracil (U) can also be present, for example, as a natural replacement for thymine when the polynucleotide is RNA. Uracil can also be used in DNA. Thus, the term “sequence” refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including natural and non-natural bases.

The term “RNA molecule” or ribonucleic acid molecule refers to a polynucleotide having a ribose sugar rather than deoxyribose sugar and typically uracil rather than thymine as one of the pyrimidine bases. An RNA molecule of the invention is generally single-stranded, but can also be double-stranded. In the context of an RNA molecule from an RNA sample, the RNA molecule can include the single-stranded molecules transcribed from DNA in the cell nucleus, mitochondrion or chloroplast, which have a linear sequence of nucleotide bases that is complementary to the DNA strand from which it is transcribed.

In some embodiments, a polynucleotide can further comprise one or more heterologous regulatory elements. For example, in some embodiments, the regulatory element is one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; or combinations thereof.

“Post-transcriptional regulatory elements” are DNA segments and/or mechanisms that affect mRNA after it has been transcribed. Mechanisms of post-transcriptional mechanisms include splicing events; capping, splicing, and addition of a Poly (A) tail, and other mechanisms known to those having ordinary skill in the art.

“Promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.

“Protein” has the same meaning as “peptide” and/or “polypeptide” in this document.

“Ratio” refers to the quantitative relation between two amounts or between two objects, which shows the relationship (in amount or quantity) between the two or more amounts, or between the two or more objects. Accordingly, in some embodiments, a ratio shows the number of times a first value contains, or is contained, within a second value.

“Reading frame” refers to one of the six possible reading frames, three in each direction, of the double stranded DNA molecule. The reading frame that is used determines which codons are used to encode amino acids within the coding sequence of a DNA molecule. In some embodiments, a reading frame is a way of dividing the sequence of nucleotides in a polynucleotide and/or nucleic acid (e.g., DNA or RNA) into a set of consecutive, non-overlapping triplets.

“Recombinant CRP” refers to refers to a non-naturally-occurring, recombinant peptide, polypeptide, or protein comprising a cystine knot (CK) architecture according to Formula (II), that is derived from a modifiable CRP that does not have the cystine knot (CK) architecture according to Formula (II). As used herein, the term “recombinant” encompasses, for example, a polypeptide that comprises one or more changes, including additions, deletions, and/or substitutions, relative to its naturally occurring counterpart, or relative to a non-naturally occurring protein that does not does not have the cystine knot (CK) architecture according to Formula (II) (e.g., a non-natural, modifiable CRP), wherein such changes were introduced, e.g., by recombinant DNA techniques. The term “recombinant” also encompasses a peptide, polypeptide, or protein that comprises, consists essentially of, or consists of: an amino acid sequence generated by humans; an artificial peptide, polypeptide, or protein; a fusion protein; and/or and a chimeric polypeptide; a nucleotide sequence generated by humans; an artificial nucleotide, polynucleotide, DNA, RNA, or gene; a polynucleotide encoding a fusion protein; and/or and a polynucleotide encoding a chimeric polypeptide. Once expressed, recombinant peptides, polypeptides, and/or proteins can be purified according to standard procedures known to one of ordinary skill in the art, e.g., including but not limited to: ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. In some embodiments, recombinant proteins may be produced by any means, including, for example, peptide, polypeptide, or protein synthesis.

“Recombinant DNA” or “rDNA” refers to DNA that is comprised of two or more different DNA segments.

“Recombinant vector” means a DNA plasmid vector into which foreign DNA has been inserted.

“Regulatory elements” refers to a genetic element that controls some aspect of the expression and/or processing of nucleic acid sequences. For example, in some embodiments, a regulatory element can be found at the transcriptional and post-transcriptional level. Regulatory elements can be cis-regulatory elements (CREs), or trans-regulatory elements (TREs). In some embodiments, a regulatory element can be one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; and/or other elements that influence gene expression, for example, in a tissue-specific manner; temporal-dependent manner; to increase or decrease expression; and/or to cause constitutive expression.

“Restriction enzyme” or “restriction endonuclease” refers to an enzyme that cleaves DNA at a specified restriction site. For example, a restriction enzyme can cleave a plasmid at an EcoRI, SacII or BstXI restriction site allowing the plasmid to be linearized, and the DNA of interest to be ligated.

“Restriction site” refers to a location on DNA comprising a sequence of 4 to 8 nucleotides, and whose sequence is recognized by a particular restriction enzyme.

“Selection gene” means a gene which confers an advantage for a genetically modified organism to grow under the selective pressure.

“Serovar” or “serotype” refers to a group of closely related microorganisms distinguished by a characteristic set of antigens. In some embodiments, a serovar is an antigenically and serologically distinct variety of microorganism

“sp.” refers to species.

“ssp.” or “subsp.” refers to subspecies.

“Subcloning” or “subcloned” refers to the process of transferring DNA from one vector to another, usually advantageous vector. For example, polynucleotide encoding a mutant DVP can be subcloned into a pLB102 plasmid subsequent to selection of yeast colonies transformed with pKLAC1 plasmids.

“SSI” is an acronym that is context dependent. In some contexts, it can refer to “site-specific integration,” which is used to refer to a sequence that will permit in vivo homologous recombination to occur at a specific site within a host organism's genome. Thus, in some embodiments, the term “site-specific integration” refers to the process directing a transgene to a target site in a host-organism's genome, allowing the integration of genes of interest into pre-selected genome locations of a host-organism. However, in other contexts, SSI can refer to “surface spraying indoors,” which is a technique of applying a variable volume sprayable volume of an insecticide onto surfaces where vectors rest, such as on walls, windows, floors and ceilings.

“STA” or “Translational stabilizing protein” or “stabilizing domain” or “stabilizing protein” (used interchangeably herein) means a peptide or protein with sufficient tertiary structure that it can accumulate in a cell without being targeted by the cellular process of protein degradation. The protein can be between 5 and 50 amino acids long. The translational stabilizing protein is coded by a DNA sequence for a protein that is operably linked with a sequence encoding an insecticidal protein or a DVP in the ORF. The operably-linked STA can either be upstream or downstream of the DVP and can have any intervening sequence between the two sequences (STA and DVP) as long as the intervening sequence does not result in a frame shift of either DNA sequence. The translational stabilizing protein can also have an activity which increases delivery of the DVP across the gut wall and into the hemolymph of the insect. Examples of a STA include, without limitation, any of the translational stabilizing proteins described, or taught by this document including GFP (Green Fluorescent Protein; SEQ ID NO:57; NCBI Accession No. P42212); GNA (SEQ ID NO: 58; NCBI Accession No. AAL07474.1); or Jun a 3, (Juniperus ashei; SEQ ID NO:59; NCBI Accession No. P81295.1).

“sta” means a nucleotide encoding a translational stabilizing protein.

“Strain” refers to a genetic variant, an isolate, a subtype, a group thereof, or a culture thereof, exhibiting phenotypic and/or genotypic traits belonging to the same lineage, distinct from those of other members of the same species. For example, in some embodiments, the term “strain” can refer to one or more yeast cells having one or more characteristics that makes them differ in some way relative to other yeast cells of their species, wherein said other yeast cells do not possess the one or more characteristics.

“Structural motif” refers to the three-dimensional arrangement of peptides and/or polypeptides, and/or the arrangement of operably linked polypeptide segments. For example, the polypeptide comprising ERSP-STA-L-DVP has an ERSP motif, an STA motif, a LINKER motif, and a DVP polypeptide motif.

“Toxin” refers to a venom and/or a poison, especially a protein or conjugated protein produced by certain animals, higher plants, and pathogenic bacteria. Generally, the term “toxin” is reserved natural products, e.g., molecules and peptides found in scorpions, spiders, snakes, poisonous mushrooms, etc., whereas the term “toxicant” is reserved for man-made products and/or artificial products e.g., man-made chemical pesticides. However, as used herein, the terms “toxin” and “toxicant” are used synonymously

“Transfection” and “transformation” both refer to the process of introducing exogenous and/or heterologous DNA or RNA (e.g., a vector containing a polynucleotide that encodes a DVP) into a host organism (e.g., a prokaryote or a eukaryote). Generally, those having ordinary skill in the art sometimes reserve the term “transformation” to describe processes where exogenous and/or heterologous DNA or RNA are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and/or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the term “transformation” and “transfection” are used synonymously, regardless of whether a process describes the introduction exogenous and/or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).

“Transgene” means a heterologous and/or exogenous DNA sequence encoding a protein which is transformed into a plant.

“Transgenic host cell” or “host cell” means a cell which is transformed with a gene and has been selected for its transgenic status via an additional selection gene.

“Transgenic plant” means a plant that has been derived from a single cell that was transformed with foreign DNA such that every cell in the plant contains that transgene.

“Transient expression system” means an Agrobacterium tumefaciens-based system which delivers DNA encoding a disarmed plant virus into a plant cell where it is expressed. The plant virus has been engineered to express a protein of interest at high concentrations, up to 40% of the TSP.

“Triple expression cassette” refers to three DVP expression cassettes contained on the same vector.

“TRBO” means a transient plant expression system using Tobacco mosaic virus with removal of the viral coating protein gene.

“Trypsin cleavage” means an in vitro assay that uses the protease enzyme trypsin (which recognizes exposed lysine and arginine amino acid residues) to separate a cleavable linker at that cleavage site. It also means the act of the trypsin enzyme cleaving that site.

“TSP” or “total soluble protein” means the total amount of protein that can be extracted from a plant tissue sample and solubilized into the extraction buffer.

“UBI” refers to ubiquitin. For example, in some embodiments, UBI can refer to a ubiquitin monomer isolated from Zea mays.

“var.” refers to varietas or variety. The term “var.” is used to indicate a taxonomic category that ranks below the species level and/or subspecies (where present). In some embodiments, the term “var.” represents members differing from others of the same subspecies or species in minor but permanent or heritable characteristics.

“Variant” or “variant sequence” or “variant peptide” refers to an amino acid sequence that possesses one or more conservative amino acid substitutions or conservative modifications. The conservative amino acid substitutions in a “variant” does not substantially diminish the activity of the variant in relation to its non-variant form. For example, in some embodiments, a “variant” possesses one or more conservative amino acid substitutions when compared to a peptide with a disclosed and/or claimed sequence, as indicated by a SEQ ID NO.

“Vector” refers to the DNA segment that accepts a heterologous polynucleotide of interest (e.g., dvp). The heterologous polynucleotide of interest is known as an “insert” or “transgene.”

“Wild type” or “WT” refer to the phenotype and/or genotype (i.e., the appearance or sequence) of an organism, polynucleotide sequence, and/or polypeptide sequence, as it is found and/or observed in its naturally occurring state or condition.

“Yeast expression vector” or “expression vector” or “vector” means a plasmid which can introduce a heterologous gene and/or expression cassette into yeast cells to be transcribed and translated.

“Yield” refers to the production of a peptide, and increased yields can mean increased amounts of production, increased rates of production, and an increased average or median yield and increased frequency at higher yields. The term “yield” when used in reference to plant crop growth and/or production, as in “yield of the plant” refers to the quality and/or quantity of biomass produced by the plant.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, solid phase and liquid nucleic acid synthesis, peptide synthesis in solution, solid phase peptide synthesis, immunology, cell culture, and formulation. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al, pp 35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; J. F. Ramalho Ortigao, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, 3. eds.), vol. 2, pp. 1-284, Academic Press, New York. 12. Wiinsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000); each of these references are incorporated herein by reference in their entireties.

Throughout this specification, unless the context requires otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

All patent applications, patents, and printed publications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. And, all patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers, or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.

Wild-Type Diguetoxins and DVPS

The American Desert Spider (Diguetia canities), also known as “the desert bush spider,” is a species of coneweb spider found in desert and semi-desert habitats in the United States. Diguetia canities produces toxins that have been shown to have an insecticidal effect, while having no effect on mammals. See Bende et al., A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a. Nat Commun. 2014 Jul. 11; 5: 4350.

One of the toxins that Diguetia canities produces is, inter alia, Mu-diguetoxin-Dc1a, (also known as μ-DGTX-Dc1a, or simply “Dc1a”). An exemplary wild-type Mu-diguetoxin-Dc1a polypeptide sequence from Diguetia canities is provided herein, having the amino acid sequence of SEQ ID NO:1 (NCBI Accession No. P49126.1).

The wild-type Dc1a polypeptide exemplified in SEQ ID NO:1 includes a signal peptide region and a propeptide region. Following polypeptide processing, the mature wild-type Dc1a polypeptide possesses an amino acid sequence of “AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLDCRCLKSGFFSSKCVCRDV” (SEQ ID NO:2). Dc1a possesses an inhibitor cystine knot (ICK) motif, along with a three-strand beta-sheet that is derived from an extended N-terminal segment, and large inter-cystine loop between residues C25 and C39. Dc1a has disulfide bond connectivity between cysteines at C12 and C25; C19 and C39; C24 and C53; and C41 and C51.

Mu-diguetoxin-Dc1a Variant Polypeptides (DVPs), or pharmaceutically acceptable salts thereof, are mutants or variants that differ from the wild-type mature Mu-diguetoxin-Dc1a (SEQ ID NO:2), e.g., in some embodiments, this variance can be an amino acid substitution, amino acid deletion/insertion, or a change to the polynucleotide encoding the wild-type Mu-diguetoxin-Dc1a. The result of this variation is a non-naturally occurring polypeptide and/or polynucleotide sequence encoding the same that possesses insecticidal activity against one or more insect species relative to the wild-type Mu-diguetoxin-Dc1a.

In some embodiments, a DVP can comprise an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K-K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁I-X₁₂-C-R-D-V, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X_nis C, F, A, T, S, M, or V; and X₁₂is V, A, or T; or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP comprises an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP comprises an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP comprises an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP comprises an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 213, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP can be a homopolymer or heteropolymer of two or more DVPs, wherein the amino acid sequence of each DVP is the same or different.

In some embodiments, a DVP can be a fused protein comprising two or more DVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each DVP may be the same or different. And, in some embodiments, the linker is cleavable inside the gut or hemolymph of an insect.

In some embodiments, the DVP can be combined with one or more additional peptides and/or produces. For example, a DVP can be part of a composition comprising a DVP as described herein, and an excipient.

In some embodiments, a DVP can be encoded by a polynucleotide. For example, a polynucleotide operable to encode a DVP, said DVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K-K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁₁-X₁₂-C-R-D-V, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X_nis C, F, A, T, S, M, or V; and X₁₂is V, A, or T, or a complementary nucleotide sequence thereof. In other embodiments, if the polynucleotide encodes a DVP wherein if X₉is G, T, A, S, M or V, or X_IIis F, A, T, S, M or V, then a disulfide bond is removed.

In yet other embodiments, the polynucleotide encodes a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, or a complementary nucleotide sequence thereof.

In yet other embodiments, the polynucleotide encodes a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a complementary nucleotide sequence thereof.

In yet other embodiments, the polynucleotide encodes a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a complementary nucleotide sequence thereof.

In yet other embodiments, the polynucleotide encodes a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 213, or 217-219, or a complementary nucleotide sequence thereof.

In some embodiments, a plant, plant tissue, plant cell, plant seed, or part thereof can comprise one or more DVPs as described herein, or a polynucleotide encoding a DVP as described herein.

In some embodiments, a DVP can be produced by a method comprising: (a) preparing a vector comprising a first expression cassette comprising a polynucleotide operable to express a DVP or complementary nucleotide sequence thereof, said DVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K—K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁₁-X₁₂-C-R-D-V, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X₁₁is C, F, A, T, S, M, or V; and X₁₂is V, A, or T; or a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable salt thereof, (b) introducing the vector into a yeast cell; and (c) growing the yeast cell in a growth medium under conditions operable to enable expression of the DVP and secretion into the growth medium. In some embodiments, if X₉is G, T, A, S, M or V, or X₁₁is F, A, T, S, M or V, then a disulfide bond is removed.

In some embodiments, the vector is a plasmid comprising an alpha-MF signal. In other embodiments, the vector is transformed into a yeast strain. For example, in some embodiments, the yeast strain is selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces. In some embodiments, the yeast strain is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris. For example, in some embodiments, the yeast strain is Kluyveromyces lactis.

In some embodiments, expression of the DVP provides a yield of: at least 70 mg/L, at least 80 mg/L, at least 90 mg/L, at least 100 mg/L, at least 110 mg/L, at least 120 mg/L, at least 130 mg/L, at least 140 mg/L, at least 150 mg/L, at least 160 mg/L, at least 170 mg/L, at least 180 mg/L, at least 190 mg/L 200 mg/L, at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, at least 1,250 mg/L, at least 1,500 mg/L, at least 1,750 mg/L, at least 2,000 mg/L, at least 2,500 mg/L, at least 3,000 mg/L, at least 3,500 mg/L, at least 4,000 mg/L, at least 4,500 mg/L, at least 5,000 mg/L, at least 5,500 mg/L, at least at least 6,000 mg/L, at least 6,500 mg/L, at least 7,000 mg/L, at least 7,500 mg/L, at least 8,000 mg/L, at least 8,500 mg/L, at least 9,000 mg/L, at least 9,500 mg/L, at least 10,000 mg/L, at least 11,000 mg/L, at least 12,000 mg/L, at least 12,500 mg/L, at least 13,000 mg/L, at least 14,000 mg/L, at least 15,000 mg/L, at least 16,000 mg/L, at least 17,000 mg/L, at least 17,500 mg/L, at least 18,000 mg/L, at least 19,000 mg/L, at least 20,000 mg/L, at least 25,000 mg/L, at least 30,000 mg/L, at least 40,000 mg/L, at least 50,000 mg/L, at least 60,000 mg/L, at least 70,000 mg/L, at least 80,000 mg/L, at least 90,000 mg/L, or at least 100,000 mg/L of DVP per liter of medium. For example, in some embodiments, expression of the DVP provides a yield of at least 100 mg/L of DVP per liter of medium.

In some embodiments, expression of the DVP in the medium results in the expression of a single DVP in the medium.

In some embodiments, expression of the DVP in the medium results in the expression of a DVP polymer comprising two or more DVP polypeptides in the medium.

In some embodiments, the vector comprises two or three expression cassettes, each expression cassette operable to encode the DVP of the first expression cassette. In some embodiments, the vector comprises two or three expression cassettes, each expression cassette operable to encode the DVP of the first expression cassette, or a DVP of a different expression cassette. In some embodiments, the expression cassette is operable to encode a DVP as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219.

Exemplary DVPs of the present invention are provided in Table 1, below.

TABLE 1

Exemplary Mu-diguetoxin-Dc1a Variant Polypeptides including

shorthand name, SEQ ID NO, and full amino acid sequence listing. Nucl. = Nucleotide.

While nucleotide sequences are provided here, the nucleic acid sequence of a

nucleic acid molecule that encodes a protein or polypeptide (e.g., a DVP) can vary

due to degeneracies.

Mu-diguetoxin-Dc1a

Nucl.

SEQ
Variant Polypeptide

SEQ ID

ID NO.
Name
Amino Acid Sequence
NO.

5
Disulfide Deletion
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
76

LDCRGLKSGFFSSKFVCRDV

6
C41T/C51A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
77

LDCRTLKSGFFSSKAVCRDV

7
C41A/C51A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
78

LDCRALKSGFFSSKAVCRDV

8
C41S/C51A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
79

LDCRSLKSGFFSSKAVCRDV

9
C41V/C51A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
80

LDCRVLKSGFFSSKAVCRDV

10
C41A/C51T
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
81

LDCRALKSGFFSSKTVCRDV

11
C41A/C51S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
82

LDCRALKSGFFSSKSVCRDV

12
C41A/C51V
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
83

LDCRALKSGFFSSKVVCRDV

13
C41T/C51S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
84

LDCRTLKSGFFSSKSVCRDV

14
C41S/C51S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
85

LDCRSLKSGFFSSKSVCRDV

15
C41T/C51A/V17A
AKDGDVEGPAGCKKYDAECDSGECCQKQYLWYKWRP
86

LDCRTLKSGFFSSKAVCRDV

16
C41T/C51A/D20A
AKDGDVEGPAGCKKYDVECASGECCQKQYLWYKWRP
87

LDCRTLKSGFFSSKAVCRDV

17
C41T/C51A/S21A
AKDGDVEGPAGCKKYDVECDAGECCQKQYLWYKWRP
88

LDCRTLKSGFFSSKAVCRDV

18
C41T/C51A/W31A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLAYKWRP
89

LDCRTLKSGFFSSKAVCRDV

19
C41T/C51A/Y32A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWAKWRP
90

LDCRTLKSGFFSSKAVCRDV

20
C41T/C51A/P36A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRA
91

LDCRTLKSGFFSSKAVCRDV

21
C41T/C51A/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
92

LACRTLKSGFFSSKAVCRDV

22
C41T/C51A/L42A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
93

LDCRTAKSGFFSSKAVCRDV

23
C41T/C51A/V52A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
94

LDCRTLKSGFFSSKAACRDV

24
C41T/C51A/W31F
AKDGDVEGPAGCKKYDVECDSGECCQKQYLFYKWRP
95

LDCRTLKSGFFSSKAVCRDV

25
C41T/C51A/Y32S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWSKWRP
96

LDCRTLKSGFFSSKAVCRDV

26
C41T/C51A/W31F/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLFSKWRA
97

Y32S/P36A
LDCRTLKSGFFSSKAVCRDV

27
C41T/C51A/D20A/
AKDGDVEGPAGCKKYDVECASGECCQKQYLWYKWRP
98

L42N
LDCRTNKSGFFSSKAVCRDV

28
C41T/C51A/D20A/
AKDGDVEGPAGCKKYDVECASGECCQKQYLWYKWRP
99

L42V
LDCRTVKSGFFSSKAVCRDV

29
C41T/C51A/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
100

LACRTLKSGFFSSKAVCRDV

30
C41T/C51A/D38K
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
101

LKCRTLKSGFFSSKAVCRDV

31
C41T/C51A/D38S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
102

LSCRTLKSGFFSSKAVCRDV

32
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
103

V52T
LACRTLKSGFFSSKATCRDV

33
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
104

V52A
LACRTLKSGFFSSKAACRDV

34
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDEECDSGECCQKQYLWYKWRP
105

V17E
LACRTLKSGFFSSKAVCRDV

35
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
106

L42V
LACRTVKSGFFSSKAVCRDV

36
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
107

L42S
LACRTSKSGFFSSKAVCRDV

37
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
108

L42E
LACRTEKSGFFSSKAVCRDV

38
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
109

L42Q
LACRTQKSGFFSSKAVCRDV

39
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECASGECCQKQYLWYKWRP
110

D20A
LACRTLKSGFFSSKAVCRDV

40
C41T/C51A/D20A/
AKDGDVEGPAGCKKYDVECASGECCQKQYLWSKWRP
111

Y32S
LDCRTLKSGFFSSKAVCRDV

41
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWSKWRP
112

Y32S
LACRTLKSGFFSSKAVCRDV

42
C41T/C51A/D20A/
AKDGDVEGPAGCKKYDVECASGECCQKQYLWSKWRP
113

D38A/Y32S
LACRTLKSGFFSSKAVCRDV

43
C41T/C51A/D20A/
AKDGDVEGPAGCKKYDVECASGECCQKQYLFSKWRA
114

W31F/Y32S/P36A
LDCRTLKSGFFSSKAVCRDV

44
D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
115

LACRCLKSGFFSSKCVCRDV

45
C41S/C51T/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
116

LACRSLKSGFFSSKTVCRDV

46
C41T/C51T/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
117

LACRTLKSGFFSSKTVCRDV

47
C41S/C51S/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
118

LACRSLKSGFFSSKSVCRDV

48
C41T/C51S/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
119

LACRTLKSGFFSSKSVCRDV

49
C41V/C51T/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
120

LACRVLKSGFFSSKTVCRDV

50
C41T/C51V/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
121

LACRTLKSGFFSSKVVCRDV

51
C41S/C51V/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
122

LACRSLKSGFFSSKVVCRDV

52
C41V/C51S/D38A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
123

LACRVLKSGFFSSKSVCRDV

53
C41S/C51S/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
124

L42V
LACRSVKSGFFSSKSVCRDV

125
C41N/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
153

L42V
LACRNVKSGFFSSKAVCRDV

126
C41D/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
154

L42V
LACRDVKSGFFSSKAVCRDV

127
C41S/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
155

L42V
LACRSVKSGFFSSKAVCRDV

128
C41M/C51A/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
156

L42V
LACRMVKSGFFSSKAVCRDV

129
C41T/C51G/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
157

L42V
LACRTVKSGFFSSKGVCRDV

130
C41T/C51D/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
158

L42V
LACRTVKSGFFSSKDVCRDV

131
C41T/C51N/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
159

L42V
LACRTVKSGFFSSKNVCRDV

132
C41T/C51Q/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
160

L42V
LACRTVKSGFFSSKQVCRDV

133
C41T/C51E/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
161

L42V
LACRTVKSGFFSSKEVCRDV

134
C41T/C51V/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
162

L42V
LACRTVKSGFFSSKVVCRDV

135
C41T/C51H/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
163

L42V
LACRTVKSGFFSSKHVCRDV

136
C41T/C51M/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
164

L42V
LACRTVKSGFFSSKMVCRDV

137
C41V/C51V/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
165

L42V
LACRVVKSGFFSSKVVCRDV

138
C41M/C51M/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
166

L42V
LACRMVKSGFFSSKMVCRDV

139
C41K/C51E/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
167

L42V
LACRDVKSGFFSSKEVCRDV

140
C41E/C51K/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
168

L42V
LACREVKSGFFSSKKVCRDV

141
C41T/C51A/D20V/
AKDGDVEGPAGCKKYDVECVSGECCQKQYLWYKWRP
169

D38A/L42V
LACRTVKSGFFSSKAVCRDV

142
C41T/C51A/D20G/
AKDGDVEGPAGCKKYDVECGSGECCQKQYLWYKWRP
170

D38A/L42V
LACRTVKSGFFSSKAVCRDV

143
C41T/C51A/D20K/
AKDGDVEGPAGCKKYDVECKSGECCQKQYLWYKWRP
171

D38A/L42V
LACRTVKSGFFSSKAVCRDV

144
C41T/C51A/D20E/
AKDGDVEGPAGCKKYDVECESGECCQKQYLWYKWRP
172

D38A/L42V
LACRTVKSGFFSSKAVCRDV

145
C41T/C51A/D20L/
AKDGDVEGPAGCKKYDVECLSGECCQKQYLWYKWRP
173

D38A/L42V
LACRTVKSGFFSSKAVCRDV

146
C41T/C51A/D20N/
AKDGDVEGPAGCKKYDVECNSGECCQKQYLWYKWRP
174

D38A/L42V
LACRTVKSGFFSSKAVCRDV

147
C41T/C51A/D20Y/
AKDGDVEGPAGCKKYDVECYSGECCQKQYLWYKWRP
175

D38A/L42V
LACRTVKSGFFSSKAVCRDV

148
C41T/C51A/S21G/
AKDGDVEGPAGCKKYDVECDGGECCQKQYLWYKWRP
176

D38A/L42V
LACRTVKSGFFSSKAVCRDV

149
C41T/C51A/E18P/
AKDGDVEGPAGCKKYDVPCDSGECCQKQYLWYKWRP
177

D38A/L42V
LACRTVKSGFFSSKAVCRDV

150
C41T/C51A/E18K/
AKDGDVEGPAGCKKYDVKCDSGECCQKQYLWYKWRP
178

D38A/L42V
LACRTVKSGFFSSKAVCRDV

151
C41T/C51A/E18S/
AKDGDVEGPAGCKKYDVSCDSGECCQKQYLWYKWRP
179

D38A/L42V
LACRTVKSGFFSSKAVCRDV

152
C41T/C51A/E18D/
AKDGDVEGPAGCKKYDVDCDSGECCQKQYLWYKWRP
180

D38A/L42V
LACRTVKSGFFSSKAVCRDV

181
C41V/C51T/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
230

L42V
LACRVLKSGFFSSKTVCRDV

182
C41N/C51A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
231

LDCRNLKSGFFSSKAVCRDV

187
Y32S/P36A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWSKWRA
232

LDCRCLKSGFFSSKCVCRDV

188
Y32K/P36A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWKKWRA
233

LDCRCLKSGFFSSKCVCRDV

189
Y32H/P36A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWHKWRA
234

LDCRCLKSGFFSSKCVCRDV

190
W31F/Y32S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLFSKWRP
235

LDCRCLKSGFFSSKCVCRDV

191
W31F/Y32S/P36A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLFSKWRA
236

LDCRCLKSGFFSSKCVCRDV

192
Y32H/P36A/C41A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWHKWRA
237

C51A
LDCRALKSGFFSSKAVCRDV

202
C41T/C51A/Y29A
AKDGDVEGPAGCKKYDVECDSGECCQKQALWYKWRP
238

LDCRTLKSGFFSSKAVCRDV

203
C41T/C51A/G45A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
239

LDCRTLKSAFFSSKAVCRDV

204
C41T/C51A/F47A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
240

LDCRTLKSGFASSKAVCRDV

205
C41T/C51A/R54A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
241

LDCRTLKSGFFSSKAVCADV

206
C41T/C51A/Y32A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWAKWRP
242

LDCRTLKSGFFSSKAVCRDV

207
C41T/C51A/P36A
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRA
243

LDCRTLKSGFFSSKAVCRDV

208
C41T/C51A/D38A/
AKDGDVEGPAGCKKYDVECASGECCQKQYLWYKWRP
244

L42H
LDCRTHKSGFFSSKAVCRDV

209
Y32S/D38A/C41S/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWSKWRP
245

L42I/C51S
LACRSIKSGFFSSKSVCRDV

210
D38A/L42I/C41S/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
220

C51S
LACRSIKSGFFSSKSVCRDV

211
K2L/D38A/C41S/
ALDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
221

C51S
LACRSLKSGFFSSKSVCRDV

212
Y32S/D38A/
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWSKWRP
222

C41S/C51S
LACRSLKSGFFSSKSVCRDV

213
K2L/Y32S/D38A/
ALDGDVEGPAGCKKYDVECDSGECCQKQYLWSKWRP
223

C41S/C51S
LACRSLKSGFFSSKSVCRDV

214
D38T/C41S/C51S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
224

LTCRSLKSGFFSSKSVCRDV

215
D38S/C41S/C51S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
225

LSCRSLKSGFFSSKSVCRDV

216
D38M/C41S/C51S
AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
226

LMCRSLKSGFFSSKSVCRDV

217
K2L/Y32S/L42I
ALDGDVEGPAGCKKYDVECDSGECCQKQYLWSKWRP
227

LDCRCIKSGFFSSKCVCRDV

218
K2L/Y32S/D38A/
ALDGDVEGPAGCKKYDVECDSGECCQKQYLWSKWRP
228

L42I/C41S/C51S
LACRSIKSGFFSSKSVCRDV

219
K2L/D38A/L42I/
ALDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRP
229

C41S/C51S
LACRSIKSGFFSSKSVCRDV

In some embodiments, a DVP can have a disulfide deletion. For example, in some embodiments, a DVP can have amino acid substitutions at residues C41 and C51, resulting in the deletion of a disulfide bond. In some embodiments, a DVP with a disulfide deletion can have an amino acid substitution of C51G, C51IF, and/or both, relative to SEQ TD NO:2. In some embodiments, a DVP with a disulfide deletion can have an amino acid sequence of SEQ TD NO: 5. In some embodiments, the term “Disulfide deletion” refers to those embodiments that have an amino acid substitution of C51G, C51IF, and/or both, relative to SEQ TD NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T and C51A relative to SEQ TD NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ TD NO:6. In some embodiments, the term “C41T/C51A” refers to those embodiments that have an amino acid substitution of C51G, C51F, and/or both, relative to SEQ ID NO: 2.

In some embodiments, a DVP can have amino acid substitutions of C41A and C51A relative to SEQ TD NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:7. In some embodiments, the term “C41A/C51A” refers to those embodiments that have an amino acid substitution of C41A and C51A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41S and C51A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:8. In some embodiments, the term “C41S/C51A” refers to those embodiments that have an amino acid substitution of C41S and C51A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41V and C51A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:9. In some embodiments, the term “C41V/C51A” refers to those embodiments that have an amino acid substitution of C41V and C51A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41A and C51T relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:10. In some embodiments, the term “C41A/C51T” refers to those embodiments that have an amino acid substitution of C41A and C51T relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41A and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:11. In some embodiments, the term “C41A/C51S” refers to those embodiments that have an amino acid substitution of C41A and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41A and C51V relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:12. In some embodiments, the term “C41A/C51V” refers to those embodiments that have an amino acid substitution of C41A and C51V relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:13. In some embodiments, the term “C41T/C51S” refers to those embodiments that have an amino acid substitution of C41T and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41S and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:14. In some embodiments, the term “C41S/C51S” refers to those embodiments that have an amino acid substitution of C41S and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and V17A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:15. In some embodiments, the term “C41T/C51A/V17A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and V17A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and D20A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:16. In some embodiments, the term “C41T/C51A/D20A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and D20A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and S21A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:17. In some embodiments, the term “C41T/C51A/S21A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and S21A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and W31A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:18. In some embodiments, the term “C41T/C51A/W31A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and W31A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and Y32A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 19. In some embodiments, the term “C41T/C51A/Y32A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and Y32A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and P36A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:20. In some embodiments, the term “C41T/C51A/P36A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and P36A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:21. In some embodiments, the term “C41T/C51A/D38A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and L42A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:22. In some embodiments, the term “C41T/C51A/L42A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and L42A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and V52A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:23. In some embodiments, the term “C41T/C51A/V52A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and V52A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and W31F relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:24. In some embodiments, the term “C41T/C51A/W31F” refers to those embodiments that have an amino acid substitution of C41T, C51A, and W31F relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and Y32S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:25. In some embodiments, the term “C41T/C51A/Y32S” refers to those embodiments that have an amino acid substitution of C41T, C51A, and Y32S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, W31F, Y32S, and P36A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:26. In some embodiments, the term “C41T/C51A/W31F/Y32S/P36A” refers to those embodiments that have an amino acid substitution of C41T, C51A, W31F, Y32S, and P36A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D20A, and L42N relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:27. In some embodiments, the term “C41T/C51A/D20A/L42N” refers to those embodiments that have an amino acid substitution of C41T, C51A, D20A, and L42N relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D20A, and L42V relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:28. In some embodiments, the term “C41T/C51A/D20A/L42V” refers to those embodiments that have an amino acid substitution of C41T, C51A, D20A, and L42V relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:29. In some embodiments, the term “C41T/C51A/D38A” refers to those embodiments that have an amino acid substitution of C41T, C51A, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and D38K relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:30. In some embodiments, the term “C41T/C51A/D38K” refers to those embodiments that have an amino acid substitution of C41T, C51A, and D38K relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, and D38S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:31. In some embodiments, the term “C41T/C51A/D38S” refers to those embodiments that have an amino acid substitution of C41T, C51A, and D38S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and V52T relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:32. In some embodiments, the term “C41T/C51A/D38A/V52T” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and V52T relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and V52A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:33. In some embodiments, the term “C41T/C51A/D38A/V52A” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and V52A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and V17E relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:34. In some embodiments, the term “C41T/C51A/D38A/V17E” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and V17E relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and L42V relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:35. In some embodiments, the term “C41T/C51A/D38A/L42V” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and L42V relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and L42S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:36. In some embodiments, the term “C41T/C51A/D38A/L42S” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and L42S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and L42E relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:37. In some embodiments, the term “C41T/C51A/D38A/L42E” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and L42E relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and L42Q relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:38. In some embodiments, the term “C41T/C51A/D38A/L42Q” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and L42Q relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and D20A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:39. In some embodiments, the term “C41T/C51A/D38A/D20A” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and D20A relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D20A, and Y32S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:40. In some embodiments, the term “C41T/C51A/D20A/Y32S” refers to those embodiments that have an amino acid substitution of C41T, C51A, D20A, and Y32S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D38A, and Y32S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:41. In some embodiments, the term “C41T/C51A/D38A/Y32S” refers to those embodiments that have an amino acid substitution of C41T, C51A, D38A, and Y32S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D20A, D38A, and Y32S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:42. In some embodiments, the term “C41T/C51A/D20A/D38A/Y32S” refers to those embodiments that have an amino acid substitution of C41T, C51A, D20A, D38A, and Y32S relative to SEQ ID NO:2.

In some embodiments, a DVP can have amino acid substitutions of C41T, C51A, D20A, W31F, Y32S, and P36A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:43. In some embodiments, the term “C41T/C51A/D20A/W31F/Y32S/P36A” refers to those embodiments that have an amino acid substitution of C41T, C51A, D20A, W31F, Y32S, and P36A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:44. In some embodiments, the term “D38A” refers to those embodiments that have an amino acid substitution of D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41S, C51T, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:45. In some embodiments, the term “C41S/C51T/D38A” refers to those embodiments that have an amino acid substitution of C41S, C51T, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41T, C51T, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:46. In some embodiments, the term “C41T/C51T/D38A” refers to those embodiments that have an amino acid substitution of C41T, C51T, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41S, C51S, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:47. In some embodiments, the term “C41S/C51S/D38A” refers to those embodiments that have an amino acid substitution of C41S, C51S, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41T, C51S, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:48. In some embodiments, the term “C41T/C51S/D38A” refers to those embodiments that have an amino acid substitution of C41T, C51S, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41V, C51T, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:49. In some embodiments, the term “C41V/C51T/D38A” refers to those embodiments that have an amino acid substitution of C41V, C51T, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41T, C51V, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:50. In some embodiments, the term “C41T/C51V/D38A” refers to those embodiments that have an amino acid substitution of C41T, C51V, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41S, C51V, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:51. In some embodiments, the term “C41S/C51V/D38A” refers to those embodiments that have an amino acid substitution of C41S, C51V, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41V, C51S, and D38A relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:52. In some embodiments, the term “C41V/C51S/D38A” refers to those embodiments that have an amino acid substitution of C41V, C51S, and D38A relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41S, C51iS, D38A, and L42V relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO:53. In some embodiments, the term “C41S/C51S/D38A/L42V” refers to those embodiments that have an amino acid substitution of C41S, C51S, D38A, and L42V relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of C41S, C51iS, D38A, and L42V relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 53. In some embodiments, the term “C41S/C51S/D38A/L42V” refers to those embodiments that have an amino acid substitution of C41S, C51S, D38A, and L42V relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of D38A, L42I, C41S, and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 210. In some embodiments, the term “D38A/L42I/C41S/C51S” refers to those embodiments that have an amino acid substitution of D38A, L42I, C41S, and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of K2L, D38A, C41S, and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 211. In some embodiments, the term “K2L/D38A/C41S/C51S” can refer to those embodiments that have an amino acid substitution of K2L, D38A, C41S, and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of Y32S, D38A, C41S, and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 212. In some embodiments, the term “Y32S/D38A/C41S/C51S” can refer to those embodiments that have an amino acid substitution of Y32S, D38A, C41S, and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of K2L, Y32S, D38A, C41S, and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 213. In some embodiments, the term “K2L/Y32S/D38A/C41S/C51S” can refer to those embodiments that have an amino acid substitution of K2L, Y32S, D38A, C41S, and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of D38T, C41S, and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 214. In some embodiments, the term “D38T/C41S/C51S” can refer to those embodiments that have an amino acid substitution of D38T, C41S, and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of D38S, C41S, and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 215. In some embodiments, the term “D38S/C41S/C51S” can refer to those embodiments that have an amino acid substitution of D38S, C41S, and C51S relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of K2L, Y32S, and L42I relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 217. In some embodiments, the term “K2L/Y32S/L42I” can refer to those embodiments that have an amino acid substitution of K2L, Y32S, and L42I relative to SEQ ID NO:2.

In some embodiments, a DVP can have an amino acid substitution of K2L, Y32S, L42I, C41S, and C51S relative to SEQ ID NO:2. For example, in some embodiments, a DVP can have an amino acid sequence of SEQ ID NO: 217. In some embodiments, the term “K2L/Y32S/L42I/C41S/C51S” can refer to those embodiments that have an amino acid substitution of K2L, Y32S, L42I, C41S, and C51S relative to SEQ ID NO:2.

In various embodiments, polynucleotides encoding DVPs can be used to transform plant cells, yeast cells, or bacteria cells. In some embodiments, the insecticidal DVP transgenic proteins may be formulated into compositions that can be sprayed or otherwise applied in any manner known to those skilled in the art to the surface of plants or parts thereof. Accordingly, DNA constructs are provided herein, operable to encode one or more DVPs under the appropriate conditions in a host cell, for example, a plant cell. Methods for controlling a pest infection by a parasitic insect of a plant cell comprises administering or introducing a polynucleotide encoding an DVP as described herein to a plant, plant tissue, or a plant cell by recombinant techniques and growing said recombinantly altered plant, plant tissue or plant cell in a field exposed to the pest. Alternatively, DVPs can be formulated into a sprayable composition consisting of a DVP and an excipient, and applied directly to susceptible plants by direct application, such that upon ingestion of the DVP by the infectious insect results in a deleterious effect.

In some embodiments, the DVP may comprise an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, the DVP may comprise an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, the DVP may comprise an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, the DVP may comprise an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 213, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, the DVP may comprise an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 128 or 147, or a pharmaceutically acceptable salt thereof.

In some embodiments, a polynucleotide operable to encode a DVP may have an nucleic acid sequence of any one of SEQ ID NOs: 77-114, 116-122, 124, 156, 158, 164, 167-168, 172, 174-175, 220-225, or 227-219. In some embodiments, the polynucleotide operable to encode a DVP may comprise a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% nucleotide sequence identity to of SEQ ID NOs: 77-114, 116-122, 124, 156, 158, 164, 167-168, 172, 174-175,220-225, or 227-219.

In some embodiments, a polynucleotide operable to encode a DVP may comprise an nucleic acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 77-114, 116-122, 124, 156, 158, 164, 167-168, 172, 174-175,220-225, or 227-219.

In some embodiments, a polynucleotide encoding a DVP can encode a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191,202-215, or 217-219.

In some embodiments, a polynucleotide of the present invention comprises a polynucleotide operable to encode a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a complementary sequence thereof.

In some embodiments, a polynucleotide of the present invention comprises a polynucleotide operable to encode a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a complementary sequence thereof.

In some embodiments, a polynucleotide of the present invention comprises a polynucleotide operable to encode a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 213, or 217-219, or a complementary sequence thereof.

In some embodiments, a polynucleotide of the present invention comprises a polynucleotide operable to encode a DVP having an amino sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, or a complementary sequence thereof.

In some embodiments, a polynucleotide of the present invention comprises a polynucleotide operable to encode a DVP having an amino sequence as set forth in any one of SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a complementary sequence thereof.

In some embodiments, a polynucleotide of the present invention comprises a polynucleotide operable to encode a DVP having an amino sequence as set forth in any one of SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a complementary sequence thereof.

In some embodiments, a polynucleotide of the present invention comprises a polynucleotide operable to encode a DVP having an amino sequence as set forth in any one of SEQ ID NOs: 213, or 217-218, or a complementary sequence thereof.

DVP-Insecticidal Proteins

In some embodiments, a DVP-insecticidal protein can be any protein, peptide, polypeptide, amino acid sequence, configuration, construct or arrangement, comprising: (1) at least one DVP, or two or more DVPs; and (2) additional non-toxin peptides, polypeptides, or proteins. For example, in some embodiments, these additional peptides, polypeptides, or proteins may have the ability to increase the mortality and/or inhibit the growth of insects exposed to the DVP-insecticidal protein, relative to the DVP alone; increase the expression of the DVP-insecticidal protein, e.g., in a host cell; and/or affect the post-translational processing of the DVP-insecticidal protein.

In some embodiments, a DVP-insecticidal protein can be a polymer comprising two or more DVPs. In yet other embodiments, a DVP-insecticidal protein can be a polymer comprising two or more DVPs, wherein the DVPs are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker.

In some embodiments, a DVP-insecticidal protein can be a polymer of amino acids that when properly folded or in its most natural thermodynamic state exerts an insecticidal activity against one or more insects. For example, in some embodiments, a DVP-insecticidal protein can be a polymer comprising two or more DVPs that are different. In other embodiments, an insecticidal protein can be a polymer of two or more DVPs that are the same.

In yet other embodiments, a DVP-insecticidal protein can comprise one or more DVPs, and one or more peptides, polypeptides, or proteins, that may assist in the DVP-insecticidal protein's folding.

In some embodiments, a DVP-insecticidal protein can comprise one or more DVPs, and one or more peptides, polypeptides, or proteins, wherein the one or more peptides, polypeptides, or proteins are protein tags that help stability or solubility. In other embodiments, the peptides, polypeptides, or proteins can be protein tags that aid in affinity purification.

In some embodiments, a DVP-insecticidal protein can refer to a one or more DVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker; one or more heterologous peptides; one or more additional polypeptides; and/or any other combination thereof. In some embodiments, an insecticidal protein can comprise a one or more DVPs as disclosed herein.

In some embodiments, a DVP-insecticidal protein can comprise a DVP homopolymer, e.g., two or more DVP monomers that are the same DVP. In some embodiments, the insecticidal protein can comprise a DVP heteropolymer, e.g., two or more DVP monomers, wherein the DVP monomers are different.

In some embodiments, the DVP-insecticidal protein may comprise a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191,202-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP-insecticidal protein can comprise one or more DVPs having an amino acid sequence set forth in SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, the DVP-insecticidal protein may comprise a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, the DVP-insecticidal protein may comprise a DVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP-insecticidal protein can comprise one or more DVPs having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191,202-215, or 217-219, or a pharmaceutically acceptable salt thereof.

In some embodiments, the DVP-insecticidal protein can comprise one or more DVPs, wherein the DVPs are the same or different.

Exemplary methods for the generation of cleavable and non-cleavable linkers can be found in U.S. patent application Ser. No. 15/727,277; and PCT Application No. PCT/US2013/030042, the disclosure of which are incorporated herein by reference in their entireties.

In some embodiments, a DVP-insecticidal protein can be a fusion protein comprising one or more DVPs as described herein, operably linked to an alpha mating factor (alpha-MF) peptide.

An “alpha mating factor (alpha-MF) peptide” or “alpha-MF signal” or “alpha-MF” or “alpha mating factor secretion signal” or “αMF secretion signal” (all used interchangeably) refers to a signal peptide that allows for secreted expression in a recombinant expression system, when the alpha-MF peptide is operably linked to a recombinant peptide of interest (e.g., a DVP). The Alpha-MF peptide directs nascent recombinant polypeptides to the secretory pathway of the recombinant expression system (e.g., a yeast recombinant expression system.

Alpha-MF peptides are well known in the art. Exemplary alpha-MF peptides are provided herein, including, without limitation: Kluyveromyces lactis alpha mating factor pre-pro secretion leader of the pKLAC1 vector (SEQ ID NO: 246); NCBI Accession No. XP_454814 (SEQ ID NO: 247); Mf(alpha)1/Mf(alpha)2 (SEQ ID NO: 248; NCBI Accession No. QEU61411.1); Mating factor alpha precursor N-terminus (SEQ ID NO: 249; NCBI Accession No. KAG0674310); and the like.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein said one or more DVPs have an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K-K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁I-X₁₂-C-R-D-V, wherein the DVP comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X_nis C, F, A, T, S, M, or V; and X₁₂is V, A, or T, or a pharmaceutically acceptable salt thereof.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein said one or more DVPs have an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence according to Formula (I): A-X₁-D-G-D-V-E-G-P-A-G-C-K—K-Y-D-X₂-E-C-X₃-X₄-G-E-C-C-Q-K-Q-Y-L-X₅-X₆-K-W-R-X₇-L-X₈-C-R-X₉-X₁₀-K-S-G-F-F-S-S-K-X₁₁-X₁₂-C-R-D-V, wherein the DVP comprises at least one amino acid substitution relative to the wild-type sequence of the diguetoxin as set forth in SEQ ID NO:2, and wherein X₁is K or L; X₂is V, A, or E; X₃is D, Y, or A; X₄is S or A; X₅is W, A, F; X₆is Y, A, S, H, or K; X₇is P or A; X₈is D, A, K, S, T or M; X₉is C, G, T, A, S, M, or V; X₁₀is L, A, N, V, S, E, I, or Q; X₁₁is C, F, A, T, S, M, or V; and X₁₂is V, A, or T, or a pharmaceutically acceptable salt thereof, wherein if X₉is G, T, A, S, M or V, or X₁₁is F, A, T, S, M or V, then a disulfide bond is removed.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the one or more DVPs comprise an amino sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the one or more DVPs comprise an amino sequence as set forth in any one of SEQ ID NOs: 6-11, 15-16, 20-22, 24-26, 29, 35, 45-48, 53, 128, 136, 139-140, 144, 146-147, 187-191, 207, 210-215, or 217-219.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the one or more DVPs comprise an amino sequence as set forth in any one of SEQ ID NOs: 47, 53, 136, 139-140, 144, 146-147, 187-191, 210-215, or 217-219.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; the one or more DVPs is a homopolymer or heteropolymer of two or more DVPs, wherein the amino acid sequence of each DVP is the same or different.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the one or more DVPs, the alpha-MF, or a combination thereof, are separated by a cleavable linker or non-cleavable linker.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the cleavable linker is cleavable inside the gut or hemolymph of an insect.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the alpha-MF peptide is an alpha-MF peptide derived from a yeast species.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the alpha-MF peptide is derived from a yeast species selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the alpha-MF peptide is derived from a yeast species that is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein the alpha-MF peptide is derived from a Kluyveromyces lactis or Kluyveromyces marxianus.

In some embodiments, the alpha-MF peptide can be an alpha-MF peptide derived from a Kluyveromyces lactis.

In some embodiments, the alpha-MF peptide can be a K. lactis α-mating factor (a-MF) secretion domain (for secreted expression).

In some embodiments, the alpha-MF peptide can having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 246-249.

In some embodiments, the alpha-MF peptide can having an amino acid sequence as set forth in any one of SEQ ID NOs: 246-249.

In some embodiments, the alpha-MF peptide can having an amino acid sequence as set forth in SEQ ID NO: 246.

In some embodiments, a fusion protein can comprise one or more DVPs having an amino sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219; wherein the one or more DVPs are operably linked to an alpha-MF peptide having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 246-249.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein there are two or more DVPs.

In some embodiments, a fusion protein can comprise one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; wherein there are two or more DVPs, wherein the DVPs and/or the alpha-MF peptide are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker.

In some embodiments, a DVP-insecticidal protein can be a fusion protein comprising one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide; and further operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker (L); and/or any other combination thereof.

Any of the DVPs described herein, can be used to produce a fusion protein comprising one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide. For example, any of the DVPs described herein can be used to produce a fusion protein comprising one or more DVPs operably linked to an alpha mating factor (alpha-MF) peptide, e.g., wherein the one or more DVPs has an amino sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 6-43, 45-51, 53, 128, 130, 136, 139-140, 144, 146-147, 187-191, 202-215, or 217-219, which are likewise described herein.

Exemplary DVPs and DVP-Insecticidal Proteins

In some embodiments, a DVP or a DVP-insecticidal protein comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLACRSLKSGFFSSKSVCRDV” (SEQ ID NO: 47), or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP or a DVP-insecticidal protein comprises, consists essentially of, or consists of, the amino acid sequence: “AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLACRSLKSGFFSSKSVCRDV” (SEQ ID NO: 47), or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP or a DVP-insecticidal protein comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLACRSVKSGFFSSKSVCRDV” (SEQ ID NO: 53), or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP or a DVP-insecticidal protein comprises, consists essentially of, or consists of, the amino acid sequence: “AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLACRSVKSGFFSSKSVCRDV” (SEQ ID NO: 53), or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP or a DVP-insecticidal protein comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLACRTVKSGFFSSKMVCRDV” (SEQ ID NO: 136), or a pharmaceutically acceptable salt thereof.

In some embodiments, a DVP or a DVP-insecticidal protein comprises, consists essentially of, or consists of, the amino acid sequence: “AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLACRTVKSGFFSSKMVCRDV” (SEQ ID NO: 136), or a pharmaceutically acceptable salt thereof.

“AKDGDVEGPAGCKKYDVECDSGECCQKQYLWYKWRPLACRDVKSGFFSSKEVCRDV”(SEQ ID NO: 139), or a pharmaceutically acceptable salt thereof.