Methods and compositions for peptide production and purification

Abstract

Provided herein are methods for producing peptides from inclusion bodies. Also provided herein are onconase variants with improved properties.

Description

[Technical Field]

[0001] Reference to the Electronic Sequence Listing The contents of the electronic sequence listing (185952001040SEQLIST.xml; size: 3,250,395 bytes; and creation date: February 7, 2024) are incorporated herein by reference in their entirety.

[0002] The present invention relates to a method for expressing peptides and purifying them from inclusion bodies.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 486,652, 63 / 486,653, and 63 / 486,654, all of which were filed on February 23, 2023, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0004] Although short peptides can be produced in high yields by chemical synthesis (Merrifield, R.B., (1993) J. Am. Chem. Soc. 85:2149-2154), recombinant production offers the possibility of large-scale production at a more reasonable cost. However, bioproduction of short peptides poses particular challenges. Peptides shorter than 30 amino acids in length are usually unstructured and therefore difficult to express solely in microbial cultures by recombinant DNA techniques. The production of short peptides by recombinant means is further complicated by potential toxicity to host cells or their high susceptibility to in vivo proteolysis, which results in low peptide yields, if any at all (Gottesman, S. (1990) Methods in Enzymology 185: 119-129; Goff and Goldberg, (1986) in Maximizing Gene Expression p287-314). Fusing short peptides to larger carrier proteins can help reduce toxicity, confer stability, and aid purification (Terpe, K. (2003) Appl. Microbiol. Biotechnol. 60:523-533), but even when the peptide is part of a larger fusion protein, high concentrations of the fusion peptide in cells can have toxic effects.

[0005] The use of an insoluble carrier protein as a fusion protein can help reduce peptide toxicity and proteolysis by promoting its aggregation into insoluble inclusion bodies. Inclusion bodies provide protection from proteolysis and serve as a purification substrate (T. Kempe et al., (1985) Gene 39:239-245). However, common methods for separating peptides from carrier proteins can be inefficient and usually require expensive cleavage reagents and affinity columns for purification. For example, protease-based recovery using site-specific proteases or autoprotease fusion partners has been used to separate peptides from carrier proteins with some success, but these strategies require large peptide fusions and are not compatible with concatemeric peptide production strategies.

[0006] Peptide production methods using onconase as an insoluble carrier protein have been developed and represent a promising strategy for producing peptides in bacteria or yeast. One of these onconase-based strategies was recently described by Pane, K. et al. (2016) PLoS One. 1 l(l):e0146552 ("Pane, et al."). Pane et al. used an onconase-peptide fusion construct to express peptides in Escherichia coli (E. coli) and purify them from inclusion bodies. Separation of the peptide from onconase was performed using a chemical cleavage strategy, which can be performed using inexpensive reagents and targets peptide bonds that are typically present infrequently in proteins. Notably, the protocol described by Pane et al. required the use of chaotropic agents but eliminated the use of chromatographic steps. This protocol still resulted in low peptide yields and purity, which could potentially compromise downstream applications of peptides produced by this method. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] EP- A-36776 [Non-licensed literature]

[0008] [Non-licensed Document 1] Merrifield, RB, (1993) J. Am. Chem. Soc. 85:2149-2154 [Non-licensed Document 2] Gottesman,S.(1990)Methods in Enzymology 185: 119-129; [Non-licensed Document 3] Goff and Goldberg, (1986) in Maximizing Gene Expression p287-314 [Non-licensed Document 4] Terpe, K. (2003) Appl. Microbiol. Biotechnol. 60:523-533 [Non-licensed Document 5] T. Kempe, (1985)Gene 39:239-245 [Non-licensed Document 6] Pane, Kら.(2016)PLoS One. 1 l(l):e0146552 [Non-licensed Document 7] Gavit, P and Better, M., J. Biotechnol., 79:127-136 (2000) [Non-licensed Document 8] Szoka, DNA, 5(1): 11-20(1986) [Non-licensed Document 9] Walker, JM, The Proteomics Protocols Handbook, 2005, Humana Press, Totowa, NJ [Non-licensed Document 10] Betton and Hofhug, J. Biol. Chem. 271:8046-8052 (1996) [Non-licensed Document 11] Cobum and Mackie, J. Biol. Chem. 271: 1048-1053 (1996) [Non-Patent Document 12] Derman and Beckwith, J Bacteriol. 177:3764-3770 (1995) [Non-Patent Document 13] Georgiou et al., Appl. Env. Microbial. 52: 1157-1161 (1986) [Non-Patent Document 14] Dekker et al., Eur. J. Biochem. 232:214-219 (1995) [Non-Patent Document 15] Rinas and Bailey, Appl. Env. Microbiol. 59:561-566 (1993) [Non-Patent Document 16] Georgiou et al., Appl.Env.Microbiol.52: 1157-1161 (1986) [Non-Patent Document 17] Schneider et al., Prot. Exp. Purif. 6: 10-14 (1995) [Non-Patent Document 18] Tokatlidis et al., FEBS Lett. 282:205-208 (1991) [Non-Patent Document 19] Oeda et al., J. Bacteriol. 171:3568-3571 (1989) [Non-Patent Document 20] Kuhelj et al., Eur. J. Biochem. 229:533-539 (1995) [Non-Patent Document 21] Vandenbroeck et al., Eur. J. Biochem. 215:481-486 (1993) [Non-Patent Document 22] Chatterjee et al., Gene 97:13-19 (1991) [Non-Patent Document 23] Hoog et al., BioSci. Rep. 4:917-923 (1984) [Non-Patent Document 24] Vinogradov, et al. (2003) Anal Biochem.15; 320(2):234-8 [Non-Patent Document 25] Ausubel et al. (1992) Current Protocols in Molecular Biology, Green / Wiley, New York, NY [Non-Patent Document 26] Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, NY [Non-Patent Document 27] Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY [Non-patent document 28] Wu (ed.) (1993) Meth. Enzymol. 218, Part I [Non-Patent Document 29] Wu (ed.) (1979) Meth. Enzymol. 68 [Non-Patent Document 30] Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101 [Non-Patent Document 31] Grossman and Moldave (eds.) Meth. Enzymol. 65 [Non-Patent Document 32] Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [Non-Patent Document 33] Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley [Non-Patent Document 34] Schleif and Wensink (1982) Practical Methods in Molecular Biology [Non-Patent Document 35] Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK. [Non-Patent Document 36] Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK [Non-Patent Document 37] Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York [Non-Patent Document 38] Ausubel et al. (1992) Current Protocols in Molecular Biology, Greene / Wiley, New York, NY [Non-Patent Document 39] Current Protocols in Molecular Biology, Vol. 2, eds. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13 (1988) [Non-Patent Document 40] Bitter et al., Expression and Secretion Vectors for Yeast, in Methods in Enzymology, eds. Wu & Grossman, 31987, Acad. Press, NY, Vol. 153, pp. 516-544 (1987) [Non-Patent Document 41] Glover, DNA Cloning, Vol. II, IRL Press, Wash., DC, Ch. 3 (1986) [Non-Patent Document 42] Bitter, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, NY, Vol. 152, pp. 673-684 (1987) [Non-Patent Document 43] The Molecular Biology of the Yeast Saccharomyces, Eds. Strathem et al., Cold Spring Harbor Press, Vols. I and II (1982) [Non-Patent Document 44] Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, ed. DM Glover, IRL Press, Wash., DC (1986) [Non-Patent Document 45] Studier and Moffatt, J. Mol. Biol. 189: 113 (1986) [Non-Patent Document 46] Chang et al., Nature 275:615, 1978 [Non-Patent Document 47] Goeddel et al., Nature 281:544 (1979) [Non-Patent Document 48] Goeddel et al., Nucl. Acids Res. 8:4057 (1980) [Non-Patent Document 49] Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; p. 412 (1982) Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there remains a need for methods for producing short peptides that avoid toxicity and proteolysis and allow for simple, inexpensive and efficient recovery of the peptides. [Means for solving the problem]

[0010] Embodiment 1: A method for producing an oligopeptide, comprising: expressing a fusion polypeptide comprising an insoluble carrier polypeptide operably linked by peptide bonds to two or more oligopeptides; and Releasing two or more oligopeptides from an insoluble carrier polypeptide by sequence-specific chemical cleavage of peptide bonds. A method comprising:

[0011] Embodiment 2: The method of embodiment 1, wherein the insoluble carrier polypeptide comprises an onconase polypeptide.

[0012] Embodiment 3: The method of embodiment 1, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

[0013] Embodiment 4: The insoluble carrier polypeptide is selected from the group consisting of a trpΔLE polypeptide, a ketosteroid isomerase (KSI) polypeptide, a β-galactosidase polypeptide, a PagP polypeptide, a truncated E. coli PurF F4 fragment polypeptide, a Pseudomonas aeruginosa PaP3.30 polypeptide, a histone fold domain of human transcription factor TAF12 (TAF12-HFD) polypeptide, a cleavable self-aggregation tag INTEIN-ELK16, an E. coli maltose binding protein, an E. coli RNAse II polypeptide, an E. coli alkaline phosphatase polypeptide, an E. coli phospholipase A polypeptide, an E. coli β-lactamase polypeptide, a Salmonella typhimurium MalK protein, a Clostridium thermocellum endoglucanase D polypeptide, a Bacillus thuringiensis polypeptide, a Bacillus sp. aizawai IPL7 insecticidal protein, a human procathepsin B polypeptide, a porcine interferon-γ polypeptide, a T5 DNA polymerase polypeptide, and an E. coli thioredoxin polypeptide.

[0014] Embodiment 5. The method of any one of embodiments 1-4, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

[0015] Embodiment 6: The method of any one of embodiments 1 to 5, wherein two or more oligopeptides are different.

[0016] Embodiment 7: The method of any one of embodiments 1 to 6, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

[0017] Embodiment 8: The method of any one of embodiments 1 to 6, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

[0018] Embodiment 9. The method of embodiment 8, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

[0019] Embodiment 10: The method of any one of embodiments 1-9, wherein (i) the peptide bond comprises a methionine and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises a tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises a cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

[0020] Embodiment 11: The method of any one of embodiments 1 to 9, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage uses acetic acid.

[0021] Embodiment 12: The method of any one of embodiments 1 to 11, wherein the oligopeptides are operably linked by peptide bonds and are released from one another when the oligopeptides are released from the insoluble carrier polypeptide.

[0022] Embodiment 13: The method of any one of embodiments 1 to 11, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the insoluble carrier polypeptide, they are released from one another by sequence-specific chemical cleavage of the different peptide bonds.

[0023] Embodiment 14: The method of embodiment 13, wherein (i) the different peptide bonds comprise methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the different peptide bonds comprise tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds, and the sequence-specific chemical cleavage uses formic acid; (iv) the different peptide bonds comprise asparagine-glycine bonds, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the different peptide bonds comprise cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0024] Embodiment 15: The method of embodiment 13, wherein the different peptide bonds comprise Asp-Pro bonds and the sequence-specific chemical cleavage uses acetic acid.

[0025] Embodiment 16: The method of any one of embodiments 1 to 15, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

[0026] Embodiment 17: The method of any one of embodiments 1 to 16, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

[0027] Embodiment 18: The method of any one of embodiments 1 to 15, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0028] Embodiment 19: The method of any one of embodiments 1 to 18, wherein the fusion peptide is expressed in bacteria or yeast.

[0029] Embodiment 20: The method of embodiment 19, wherein the bacterium is Escherichia coli or Vibrio natriegens.

[0030] Embodiment 21: The method of embodiment 19 or embodiment 20, wherein the yield of released oligopeptide is 10 mg per liter of bacterial culture, 20 mg per liter of bacterial culture, 30 mg per liter of bacterial culture, 40 mg per liter of bacterial culture, 50 mg per liter of bacterial culture, at least 1 g per liter, or at least 5 g per liter.

[0031] Embodiment 22: A fusion polypeptide comprising an insoluble carrier polypeptide operably linked to two or more oligopeptides, wherein the operable linkages between the two or more oligopeptides and the insoluble carrier polypeptide comprise peptide bonds capable of sequence-specific chemical cleavage.

[0032] Embodiment 23: The fusion polypeptide of embodiment 22, wherein the insoluble carrier polypeptide comprises an onconase polypeptide.

[0033] Embodiment 24: The fusion polypeptide of embodiment 22, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

[0034] Embodiment 25: The insoluble carrier polypeptide is selected from the group consisting of a trpΔLE polypeptide, a ketosteroid isomerase (KSI) polypeptide, a β-galactosidase polypeptide, a PagP polypeptide, a truncated Escherichia coli PurF F4 fragment polypeptide, a Pseudomonas aeruginosa PaP3.30 polypeptide, a histone fold domain of human transcription factor TAF12 (TAF12-HFD) polypeptide, a cleavable self-aggregation tag INTEIN-ELK16, an Escherichia coli maltose binding protein, an Escherichia coli RNAse II polypeptide, an Escherichia coli alkaline phosphatase polypeptide, an Escherichia coli phospholipase A polypeptide, an Escherichia coli β-lactamase polypeptide, a Salmonella typhimurium MalK protein, a Clostridium thermocellum endoglucanase D polypeptide, a Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, a human procathepsin B polypeptide, a porcine interferon-γ polypeptide, a T5 23. The fusion polypeptide of embodiment 22, comprising a DNA polymerase polypeptide and an E. coli thioredoxin polypeptide.

[0035] Embodiment 26. The fusion polypeptide of any one of embodiments 22-25, comprising three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

[0036] Embodiment 27: The fusion polypeptide of any one of embodiments 22 to 26, wherein two or more oligopeptides are different.

[0037] Embodiment 28: The fusion polypeptide of any one of embodiments 22 to 27, wherein the oligopeptides are all operably linked to the N-terminus of the insoluble carrier polypeptide or the oligopeptides are all operably linked to the C-terminus of the insoluble carrier polypeptide.

[0038] Embodiment 29: The fusion polypeptide of any one of embodiments 22 to 27, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

[0039] Embodiment 30. The fusion polypeptide of embodiment 29, comprising two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

[0040] Embodiment 31: The fusion polypeptide of any one of embodiments 22 to 30, wherein (i) the peptide bond comprises a methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises a tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises a cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

[0041] Embodiment 32: The fusion polypeptide of any one of embodiments 22 to 30, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage uses acetic acid.

[0042] Embodiment 33: The fusion polypeptide of any one of embodiments 22 to 32, wherein the oligopeptides are operably linked by a peptide bond and can be released from one another using sequence-specific chemical cleavage.

[0043] Embodiment 34: The fusion polypeptide of any one of embodiments 22 to 32, wherein the oligopeptides are operably linked by different peptide bonds and can be released from one another by different sequence-specific chemical cleavages.

[0044] Embodiment 35: The fusion polypeptide of embodiment 34, wherein (i) the different peptide bonds comprise methionines and the different sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophans and the different sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the different sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the different sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteines and the different sequence-specific chemical cleavage uses NTCB.

[0045] Embodiment 36: The fusion polypeptide of embodiment 34, wherein the different peptide bonds comprise Asp-Pro bonds and the different sequence-specific chemical cleavage uses acetic acid.

[0046] Embodiment 37: The fusion polypeptide of any one of embodiments 22 to 36, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

[0047] Embodiment 38: The fusion polypeptide of any one of embodiments 22 to 37, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

[0048] Embodiment 39: The fusion polypeptide of any one of embodiments 22 to 36, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0049] Embodiment 40: A method for releasing an oligopeptide fused to an insoluble carrier polypeptide that forms inclusion bodies in a cell, comprising: a) expressing a fusion polypeptide comprising an oligopeptide operably linked to an insoluble carrier polypeptide, wherein the operably linked peptide bond is capable of sequence-specific chemical cleavage with acetic acid; b) purifying the inclusion bodies, and c) incubating the inclusion bodies with acid for at least 1 hour at a temperature greater than 50°C, wherein the oligopeptide is released from the fusion polypeptide by sequence-specific cleavage of peptide bonds. A method comprising:

[0050] Embodiment 41: The method of embodiment 40, wherein the insoluble carrier polypeptide comprises an onconase polypeptide.

[0051] Embodiment 42: The method of embodiment 40, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

[0052] Embodiment 43: The insoluble carrier polypeptide is selected from the group consisting of a trpΔLE polypeptide, a ketosteroid isomerase (KSI) polypeptide, a β-galactosidase polypeptide, a PagP polypeptide, a truncated Escherichia coli PurF F4 fragment polypeptide, a Pseudomonas aeruginosa PaP3.30 polypeptide, a histone fold domain of human transcription factor TAF12 (TAF12-HFD) polypeptide, a cleavable self-aggregation tag INTEIN-ELK16, an Escherichia coli maltose binding protein, an Escherichia coli RNAse II polypeptide, an Escherichia coli alkaline phosphatase polypeptide, an Escherichia coli phospholipase A polypeptide, an Escherichia coli β-lactamase polypeptide, a Salmonella typhimurium MalK protein, a Clostridium thermocellum endoglucanase D polypeptide, a Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, a human procathepsin B polypeptide, a porcine interferon-γ polypeptide, a T5 41. The method of embodiment 40, comprising a DNA polymerase polypeptide and an E. coli thioredoxin polypeptide.

[0053] Embodiment 44: The method of any one of embodiments 40 to 43, wherein the temperature in step c) is greater than 60°C, greater than 70°C, greater than 80°C, or greater than 90°C.

[0054] Embodiment 45: The method of any one of embodiments 40 to 44, wherein the temperature in step c) is less than 100°C, less than 95°C, less than 90°C, or less than 85°C.

[0055] Embodiment 46: The method of any one of embodiments 40 to 45, wherein the pH in step c) is below 3.0.

[0056] Embodiment 47: The method of any one of embodiments 40 to 45, wherein the pH in step c) is 2.6 to 2.8.

[0057] Embodiment 48: The method of any one of embodiments 40 to 47, wherein the acid is a strong acid optionally selected from hydrochloric acid and sulfuric acid.

[0058] Embodiment 49: The method of any one of embodiments 40 to 47, wherein the acid is a weak acid.

[0059] Embodiment 50: The method of embodiment 49, wherein the weak acid is acetic acid.

[0060] Embodiment 51: The method of embodiment 50, wherein the acetic acid concentration is at least 2 weight percent, at least 3 weight percent, at least 4 weight percent, or at least 5 weight percent.

[0061] Embodiment 52: The method of embodiment 50 or embodiment 51, wherein the acetic acid concentration is less than 50 weight percent, less than 45 weight percent, less than 40 weight percent, less than 35 weight percent, or less than 30 weight percent.

[0062] Embodiment 53: The method of any one of embodiments 40 to 52, wherein the insoluble carrier polypeptide is solubilized by incubating in step c).

[0063] Embodiment 54 The method of any one of embodiments 40 to 52, wherein the insoluble carrier polypeptide is not solubilized by the incubation in step c).

[0064] Embodiment 55. The method of any one of embodiments 40 to 54, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, or twenty or more oligopeptides.

[0065] Embodiment 56: The method of any one of embodiments 40 to 55, wherein two or more oligopeptides are different.

[0066] Embodiment 57: The method of any one of embodiments 40 to 56, wherein the oligopeptides are all operably linked to the N-terminus of the insoluble carrier polypeptide or the oligopeptides are all operably linked to the C-terminus of the insoluble carrier polypeptide.

[0067] Embodiment 58: The method of any one of embodiments 40 to 56, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

[0068] Embodiment 59. The method of embodiment 58, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

[0069] Embodiment 60: The method of any one of embodiments 40 to 59, wherein (i) the peptide bond comprises a methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises a tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond, and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises a cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0070] Embodiment 61: The method of any one of embodiments 40 to 60, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is acid cleavage.

[0071] Embodiment 62: The method of any one of embodiments 40 to 61, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

[0072] Embodiment 63: The method of any one of embodiments 40 to 62, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the insoluble carrier polypeptide, they are released from one another by sequence-specific chemical cleavage of the different peptide bonds.

[0073] Embodiment 64: The method of embodiment 63, wherein (i) the different peptide bonds comprise methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the different peptide bonds comprise tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds, and the sequence-specific chemical cleavage uses formic acid; (iv) the different peptide bonds comprise asparagine-glycine bonds, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the different peptide bonds comprise cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0074] Embodiment 65: The method of embodiment 64, wherein the different peptide bonds comprise Asp-Pro bonds and the sequence-specific chemical cleavage is acid cleavage.

[0075] Embodiment 66: The method of any one of embodiments 40 to 65, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

[0076] Embodiment 67: The method of any one of embodiments 40 to 66, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

[0077] Embodiment 68: The method of any one of embodiments 40 to 65, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0078] Embodiment 69: The method of any one of embodiments 40 to 68, wherein the fusion peptide is expressed in bacteria or yeast.

[0079] Aspect 70: The method of aspect 69, wherein the bacterium is Escherichia coli or Vibrio natrigens.

[0080] Embodiment 71: A method for purifying inclusion bodies containing a fusion protein, comprising: a) expressing a fusion protein comprising an oligopeptide operably linked to an insoluble carrier polypeptide that forms inclusion bodies in cells, wherein the operably linkage is a chemically cleavable amino acid sequence; b) lysing the cells to form a cell lysate; c) centrifuging the cell lysate to form a pellet; d) washing the pellet at least once, at least twice, or at least three times with a detergent buffer containing a non-ionic detergent; e) washing the pellet at least once, at least twice, or at least three times with a salt buffer containing at least 0.5 M NaCl; f) washing the pellet with water at least once, at least twice, or at least three times, thereby producing purified inclusion bodies. A method comprising:

[0081] Embodiment 72: The method of embodiment 71, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

[0082] Embodiment 73: The method of embodiment 71, wherein the insoluble carrier polypeptide comprises an onconase polypeptide.

[0083] Embodiment 74: The insoluble carrier polypeptide is selected from the group consisting of a trpΔLE polypeptide, a ketosteroid isomerase (KSI) polypeptide, a β-galactosidase polypeptide, a PagP polypeptide, a truncated Escherichia coli PurF F4 fragment polypeptide, a Pseudomonas aeruginosa PaP3.30 polypeptide, a histone fold domain of human transcription factor TAF12 (TAF12-HFD) polypeptide, a cleavable self-aggregation tag INTEIN-ELK16, an Escherichia coli maltose binding protein, an Escherichia coli RNAse II polypeptide, an Escherichia coli alkaline phosphatase polypeptide, an Escherichia coli phospholipase A polypeptide, an Escherichia coli β-lactamase polypeptide, a Salmonella typhimurium MalK protein, a Clostridium thermocellum endoglucanase D polypeptide, a Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, a human procathepsin B polypeptide, a porcine interferon-γ polypeptide, a T5 72. The method of embodiment 71, comprising a DNA polymerase polypeptide and an E. coli thioredoxin polypeptide.

[0084] Embodiment 75: The method of any one of embodiments 71 to 74, wherein the salt buffer is at least 0.6 M NaCl, at least 0.7 M NaCl, or at least 0.75 M NaCl.

[0085] Embodiment 76. The method of any one of embodiments 71 to 75, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, or twenty or more oligopeptides.

[0086] Embodiment 77: The method of any one of embodiments 71 to 76, wherein two or more oligopeptides are different.

[0087] Embodiment 78: The method of any one of embodiments 71 to 77, wherein the oligopeptides are all operably linked to the N-terminus of the insoluble carrier polypeptide or the oligopeptides are all operably linked to the C-terminus of the insoluble carrier polypeptide.

[0088] Embodiment 79: The method of any one of embodiments 71 to 77, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

[0089] Embodiment 80. The method of embodiment 79, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

[0090] Embodiment 81: The method of any one of embodiments 71 to 80, wherein (i) the peptide bond comprises a methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises a tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond, and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises a cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0091] Embodiment 82: The method of any one of embodiments 71 to 80, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is acid cleavage.

[0092] Embodiment 83 The method of any one of embodiments 71 to 82, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

[0093] Embodiment 84: The method of any one of embodiments 71 to 82, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the insoluble carrier polypeptide, they are released from one another by sequence-specific chemical cleavage of the different peptide bonds.

[0094] Embodiment 85: The method of embodiment 84, wherein (i) the different peptide bonds comprise methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the different peptide bonds comprise tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds, and the sequence-specific chemical cleavage uses formic acid; (iv) the different peptide bonds comprise asparagine-glycine bonds, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the different peptide bonds comprise cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0095] Embodiment 86: The method of embodiment 84, wherein the different peptide bonds comprise Asp-Pro bonds and the sequence-specific chemical cleavage uses acetic acid.

[0096] Embodiment 87: The method of any one of embodiments 71 to 86, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

[0097] Embodiment 88: The method of any one of embodiments 71 to 87, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

[0098] Embodiment 89: The method of any one of embodiments 71 to 86, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0099] Embodiment 90: The method of any one of embodiments 71 to 89, wherein the fusion peptide is expressed in bacteria or yeast.

[0100] Aspect 91: The method of aspect 90, wherein the bacterium is Escherichia coli or Vibrio natrigens.

[0101] Embodiment 92: A method for producing a fusion polypeptide, comprising: expressing a fusion polypeptide comprising an oligopeptide operably linked to the C-terminus of an onconase polypeptide, wherein the onconase polypeptide comprises one or more amino acid substitutions at the 11 N-terminal amino acids compared to SEQ ID NO: 1, and wherein the onconase-oligopeptide fusion protein comprising the onconase polypeptide is expressed at a higher level than a fusion protein comprising the onconase of SEQ ID NO: 1 when expressed under the same conditions.

[0102] Embodiment 93: The method of embodiment 92, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

[0103] Embodiment 94. The method of embodiment 92 or embodiment 93, wherein two or more oligopeptides are different.

[0104] Embodiment 95: The method of any one of embodiments 92 to 94, wherein the oligopeptides are all operably linked to the N-terminus of the onconase polypeptide or the oligopeptides are all operably linked to the C-terminus of the onconase polypeptide.

[0105] Embodiment 96: The method of any one of embodiments 92 to 94, wherein at least one oligopeptide is operably linked to the N-terminus of the onconase polypeptide and at least one oligopeptide is operably linked to the C-terminus of the onconase polypeptide.

[0106] Embodiment 97. The method of embodiment 96, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the onconase polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the onconase polypeptide.

[0107] Embodiment 98: The method of any one of embodiments 92 to 97, wherein (i) the peptide bond comprises a methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises a tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond, and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises a cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0108] Embodiment 99: The method of any one of embodiments 92 to 97, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage uses acetic acid.

[0109] Embodiment 100: The method of any one of embodiments 92 to 99, wherein the oligopeptides are operably linked by a peptide bond and are released from each other when the oligopeptides are released from onconase.

[0110] Embodiment 101: The method of any one of embodiments 92 to 99, wherein the oligopeptides are operably linked by different peptide bonds and are released from one another after the oligopeptides are released from onconase by sequence-specific chemical cleavage of the different peptide bonds.

[0111] Embodiment 102: The method of embodiment 101, wherein (i) the different peptide bonds comprise methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the different peptide bonds comprise tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds, and the sequence-specific chemical cleavage uses formic acid; (iv) the different peptide bonds comprise asparagine-glycine bonds, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the different peptide bonds comprise cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0112] Embodiment 103: The method of embodiment 101, wherein the different peptide bonds comprise an Asp-Pro bond and the sequence-specific chemical cleavage is an acid cleavage.

[0113] Embodiment 104: The method of any one of embodiments 92 to 103, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

[0114] Embodiment 105: The method of any one of embodiments 92 to 104, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

[0115] Embodiment 106: The method of any one of embodiments 92 to 103, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0116] Embodiment 107: The method of any one of embodiments 92 to 106, wherein the fusion peptide is expressed in bacteria or yeast.

[0117] Aspect 108: The method of aspect 107, wherein the bacterium is Escherichia coli or Vibrio natrigens.

[0118] Embodiment 109: An onconase polypeptide comprising one or more amino acid substitutions at the 11 N-terminal amino acids of the onconase polypeptide when compared to SEQ ID NO: 1, wherein the onconase polypeptide is expressed at a higher level than the onconase protein of SEQ ID NO: 1 when expressed under the same conditions.

[0119] Embodiment 110: The onconase polypeptide of embodiment 109, comprising an amino acid sequence of one of SEQ ID NOs: 2 to 10 and 15 to 22, and optionally having one, two, three, four, five or all six C-terminal histidine residues deleted as shown in Figure 20A.

[0120] Embodiment 111: A fusion polypeptide comprising an onconase polypeptide operably linked to one or more oligopeptides, wherein the operable linkage between the one or more oligopeptides and the onconase polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage, and wherein the onconase polypeptide comprises one or more amino acid substitutions in the 11 N-terminal amino acids of the onconase polypeptide compared to SEQ ID NO: 1, and wherein the onconase polypeptide is expressed at a higher level than the onconase protein of SEQ ID NO: 1 when expressed under the same conditions.

[0121] Embodiment 112: The fusion polypeptide of embodiment 111, wherein the onconase polypeptide comprises the amino acid sequence of one of SEQ ID NOs: 2-10 and 15-22, and optionally has one, two, three, four, five or all six C-terminal histidine residues deleted as shown in Figure 20A.

[0122] Embodiment 113. The fusion polypeptide of embodiment 111 or embodiment 112, comprising 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

[0123] Embodiment 114: The fusion polypeptide of any one of embodiments 111 to 113, wherein two or more oligopeptides are different.

[0124] Embodiment 115: The fusion polypeptide of any one of embodiments 111 to 114, wherein the oligopeptides are all operably linked to the N-terminus of the onconase polypeptide or the oligopeptides are all operably linked to the C-terminus of the onconase polypeptide.

[0125] Embodiment 116: The fusion polypeptide of any one of embodiments 111 to 114, wherein at least one oligopeptide is operably linked to the N-terminus of the onconase polypeptide and at least one oligopeptide is operably linked to the C-terminus of the onconase polypeptide.

[0126] Embodiment 117. The fusion polypeptide of embodiment 116, comprising two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the onconase polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the onconase polypeptide.

[0127] Embodiment 118: The fusion polypeptide of any one of embodiments 111 to 117, wherein (i) the peptide bond comprises a methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises a tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond, and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises a cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0128] Embodiment 119: The fusion polypeptide of any one of embodiments 111 to 117, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage uses acid cleavage.

[0129] Embodiment 120: The fusion polypeptide of any one of embodiments 111 to 119, wherein the oligopeptides are operably linked by a peptide bond and can be released from one another using sequence-specific chemical cleavage.

[0130] Embodiment 121: The fusion polypeptide of any one of embodiments 105 to 119, wherein the oligopeptides are operably linked by different peptide bonds and can be released from one another by different sequence-specific chemical cleavages.

[0131] Embodiment 122: The fusion polypeptide of embodiment 121, wherein (i) the different peptide bonds comprise methionine, and the different sequence-specific chemical cleavage uses cyanogen bromide; (ii) the different peptide bonds comprise tryptophan, and the different sequence-specific chemical cleavage uses BNPS-skatole; (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds, and the different sequence-specific chemical cleavage uses formic acid; (iv) the different peptide bonds comprise asparagine-glycine bonds, and the different sequence-specific chemical cleavage uses hydroxylamine; or (v) the different peptide bonds comprise cysteine, and the different sequence-specific chemical cleavage uses NTCB.

[0132] Embodiment 123: The fusion polypeptide of embodiment 121, wherein the different peptide bonds comprise an Asp-Pro bond and the different sequence-specific chemical cleavage is an acid cleavage.

[0133] Embodiment 124: The fusion polypeptide of any one of embodiments 111 to 123, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

[0134] Embodiment 125: The fusion polypeptide of any one of embodiments 111 to 124, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

[0135] Embodiment 126: The fusion polypeptide of any one of embodiments 111 to 125, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0136] Embodiment 127. An oligopeptide comprising an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid, wherein the active amino acid sequence is a miPEP and the oligopeptide regulates a miRNA; or the active amino acid sequence is a peptide microbe inhibitor and the oligopeptide inhibits a microbe.

[0137] Embodiment 128: A nucleic acid encoding the fusion polypeptide of any one of embodiments 22 to 39, or the onconase of embodiment 109 or embodiment 110, or the fusion polypeptide of any one of embodiments 111 to 126.

[0138] Embodiment 129: A cell comprising the nucleic acid of embodiment 128.

[0139] Embodiment 130: The cell of embodiment 129, which is a bacterial cell or a yeast cell.

[0140] Aspect 131: The cell of aspect 129, wherein the bacterium is Escherichia coli or Vibrio natrigens.

[0141] Embodiment 132: The cell of embodiment 131, which is a BL21 bacterial cell.

[0142] Embodiment 133: The cell of embodiment 131, which does not express the Lon and ompT proteases.

[0143] Embodiment 134: The nucleic acid of embodiment 128, which is an isolated nucleic acid.

[0144] Embodiment 135 The method of any one of embodiments 1 to 108, wherein the fusion polypeptide or fusion peptide is expressed in E. coli.

[0145] Embodiment 136: The method of any one of embodiments 1 to 108 and 135, wherein the fusion polypeptide or fusion protein is expressed in cells grown in a fermentation bioreactor.

[0146] Embodiment 137: The method of any one of embodiments 1 to 108, wherein the cleavage is carried out at a pH of about 2 to 3.5 and a temperature of about 70 to 90° C. for about 1 to 24 hours.

[0147] Embodiment 138: A method for producing a fusion polypeptide, comprising expressing a fusion polypeptide comprising an oligopeptide operably linked to the N-terminus or C-terminus of a modified TAF polypeptide, wherein the modified TAF polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23.

[0148] Embodiment 139: The method of embodiment 138, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

[0149] Embodiment 140: The method of embodiment 138 or embodiment 139, wherein two or more oligopeptides are different.

[0150] Embodiment 141: The method of any one of embodiments 138 to 140, wherein the oligopeptides are all operably linked to the N-terminus of the modified TAF polypeptide or the oligopeptides are all operably linked to the C-terminus of the modified TAF polypeptide.

[0151] Embodiment 142: The method of any one of embodiments 138 to 140, wherein at least one oligopeptide is operably linked to the N-terminus of the modified TAF polypeptide and at least one oligopeptide is operably linked to the C-terminus of the modified TAF polypeptide.

[0152] Embodiment 143 The method of embodiment 142, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the modified TAF polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the modified TAF polypeptide.

[0153] Embodiment 144: The method of any one of embodiments 138 to 143, wherein (i) the peptide bond comprises a methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises a tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond, and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises a cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0154] Embodiment 145: The method of any one of embodiments 138 to 143, wherein the peptide bond comprises an Asp-Pro bond, and the sequence-specific chemical cleavage uses acetic acid or uses sulfuric acid.

[0155] Embodiment 146: The method of any one of embodiments 138 to 145, wherein the oligopeptides are operably linked by a peptide bond and are released from each other when the oligopeptides are released from the modified TAF polypeptide.

[0156] Embodiment 147: The method of any one of embodiments 138 to 145, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the modified TAF polypeptide, they are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

[0157] Embodiment 148: The method of embodiment 147, wherein (i) the different peptide bonds comprise methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the different peptide bonds comprise tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds, and the sequence-specific chemical cleavage uses formic acid; (iv) the different peptide bonds comprise asparagine-glycine bonds, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the different peptide bonds comprise cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0158] Embodiment 149: The method of embodiment 147, wherein the different peptide bonds comprise an Asp-Pro bond and the sequence-specific chemical cleavage is acid cleavage.

[0159] Embodiment 150: The method of any one of embodiments 138 to 149, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

[0160] Embodiment 151: The method of any one of embodiments 138 to 150, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

[0161] Embodiment 152: The method of any one of embodiments 138 to 149, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0162] Embodiment 153: The method of any one of embodiments 138 to 152, wherein the fusion peptide is expressed in bacteria or yeast.

[0163] Aspect 154: The method of aspect 153, wherein the bacterium is Escherichia coli or Vibrio natrigens.

[0164] Embodiment 155: A modified TAF polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23.

[0165] Embodiment 156: The modified TAF polypeptide of embodiment 155, wherein the modified TAF polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

[0166] Embodiment 157: A fusion polypeptide comprising a modified TAF polypeptide operably linked to one or more oligopeptides, wherein the operable linkage between the one or more oligopeptides and the modified TAF polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage, and wherein the modified TAF polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23.

[0167] Embodiment 158: The fusion polypeptide of embodiment 157, wherein the modified TAF polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

[0168] Embodiment 159. The fusion polypeptide of embodiment 157 or embodiment 158, comprising three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

[0169] Embodiment 160: The fusion polypeptide of any one of embodiments 157 to 159, wherein two or more oligopeptides are different.

[0170] Embodiment 161: The fusion polypeptide of any one of embodiments 157 to 160, wherein the oligopeptides are all operably linked to the N-terminus of the modified TAF polypeptide or the oligopeptides are all operably linked to the C-terminus of the modified TAF polypeptide.

[0171] Embodiment 162: The fusion polypeptide of any one of embodiments 157 to 160, wherein at least one oligopeptide is operably linked to the N-terminus of the modified TAF polypeptide and at least one oligopeptide is operably linked to the C-terminus of the modified TAF polypeptide.

[0172] Embodiment 163. The fusion polypeptide of embodiment 162, comprising two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the modified TAF polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the modified TAF polypeptide.

[0173] Embodiment 164: The fusion polypeptide of any one of embodiments 157 to 163, wherein (i) the peptide bond comprises a methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises a tryptophan, and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond, and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises a cysteine, and the sequence-specific chemical cleavage uses NTCB.

[0174] Embodiment 165: The fusion polypeptide of any one of embodiments 157 to 163, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is acid cleavage.

[0175] Embodiment 166: The fusion polypeptide of any one of embodiments 157 to 165, wherein the oligopeptides are operably linked by a peptide bond and can be released from one another using sequence-specific chemical cleavage.

[0176] Embodiment 167: The fusion polypeptide of any one of embodiments 157 to 165, wherein the oligopeptides are operably linked by different peptide bonds and can be released from each other by different sequence-specific chemical cleavages.

[0177] Embodiment 168: The fusion polypeptide of embodiment 167, wherein (i) the different peptide bonds comprise methionine and the different sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophan and the different sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the different sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the different sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteine ​​and the different sequence-specific chemical cleavage uses NTCB.

[0178] Embodiment 169: The fusion polypeptide of embodiment 167, wherein the different peptide bond comprises an Asp-Pro bond and the different sequence-specific chemical cleavage is acid cleavage.

[0179] Embodiment 170: The fusion polypeptide of any one of embodiments 157 to 169, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

[0180] Embodiment 171: The fusion polypeptide of any one of embodiments 157 to 170, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

[0181] Embodiment 172: The fusion polypeptide of any one of embodiments 157 to 171, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0182] Embodiment 173: A nucleic acid encoding the fusion polypeptide of any one of embodiments 22 to 39, or the modified TAF polypeptide of embodiment 155 or embodiment 156, or the fusion polypeptide of any one of embodiments 157 to 172.

[0183] Embodiment 174: A cell comprising the nucleic acid of embodiment 173.

[0184] Embodiment 175: The cell of embodiment 174, which is a bacterial cell or a yeast cell.

[0185] Aspect 176: The cell of aspect 174, wherein the bacterium is Escherichia coli or Vibrio natrigens.

[0186] Embodiment 177: The cell of embodiment 176, which is a BL21 bacterial cell.

[0187] Embodiment 178: The cell of embodiment 176, which does not express the Lon and ompT proteases.

[0188] Embodiment 179: The nucleic acid of embodiment 173, which is an isolated nucleic acid. [Brief explanation of the drawings]

[0189] [Figure 1] (FIG. 1A) Schematic diagram of the onconase-based fusion construct used for peptide bioproduction strategy as a representative tool for peptide production using an insoluble carrier polypeptide. (FIG. 1B) RP-HPLC chromatograms for the four pHs tested in the first experiment of Example 1, with overlapping peaks for peptide and protein fractions highlighted. [Figure 2] Figure 2A is a graph comparing the amount of peptide and fusion protein released for four pHs tested, from pH 2 to pH 5. Figure 2A shows the amount of peptide released by cleavage at the four pHs tested. Figure 2B shows the amount of fusion protein solubilized at the four pHs tested. [Figure 3] Figure 3A is a graph comparing the amount of peptide and fusion protein released for seven pHs tested, ranging from pH 1.5 to pH 3. Figure 3A shows the amount of peptide released by cleavage at the seven pHs tested. Figure 3B shows the amount of fusion protein solubilized at the seven pHs tested. [Figure 4] Figure 4A shows graphs depicting increasing amounts of the acidic form of a peptide with decreasing pH. Figure 4A shows overlaid RP-HPLC chromatograms for each of the five pHs tested with the base form on the left and the acidic form on the right. Figure 4B shows the relative amounts of the base form (black bars) and the acidic form (gray bars) at the eight pHs tested. [Figure 5]Figure 5 shows SDS PAGE of the fusion protein constructs tested and post-expression cell lysates for each. Figure 5A shows the five constructs tested. Figure 5B shows SDS PAGE with, from left to right: molecular weight markers, Onconase fusion, KSI fusion, PurF fusion, TAF12 fusion, and OmpX fusion. After cleavage and purification, the amounts of peptides (relative to Onconase as 100%) were: KSI fusion (120%), PurF fusion (75%), TAF12 fusion (265%), and OmpX fusion (37%). [Figure 6AB] Figure 6A is a schematic diagram of different onconase-based fusion strategies for peptide bioproduction as a representative tool for producing peptides using insoluble carrier polypeptides. Figure 6A is a schematic diagram of a homoconcatemer onconase fusion construct, in which several identical peptide sequences are added along with an additional aspartic acid-proline (DP) cleavage site and up to 10 copies of the peptide (N). Figure 6B is a schematic diagram of a heteroconcatemer construct, in which copies of a peptide of different sequence (M) are linked with an Asp-Pro (DP) cleavage site fused to the onconase protein. [Figure 6C] Figure 6A shows a schematic diagram of different onconase-based fusion strategies for peptide bioproduction as a representative tool for producing peptides using insoluble carrier polypeptides. Figure 6B shows an onconase fusion with a single peptide sequence at the C-terminus. [Figure 7] Figure 1 depicts SDS-PAGE analysis of the insoluble fractions of different onconase constructs with one (XI) or three (X3) peptide copies. Peptides A, B, and C are all 10 amino acid long hydrophilic peptides, but differ in their amino acid sequence. MW: molecular weight marker. [Figure 8] Figure 8 shows cleavage from a concatemer ONC fusion protein. Figure 8A shows an RP-HPLC chromatogram after 4 hours of cleavage, with the mono-, di-, and tripeptide peaks labeled. Figure 8B shows an RP-HPLC chromatogram after 16 hours of cleavage, with the mono- and dipeptide peaks labeled. [Figure 9AB]

[0033] Figure 9 depicts N-terminal variants of Onconase and the results of comparative peptide yield studies. Figure 9A is a schematic diagram of Onconase constructs (constructs A (SEQ ID NO:2403), B (SEQ ID NO:2404), C (SEQ ID NO:2405), D (SEQ ID NO:2406), E (SEQ ID NO:2407), F (SEQ ID NO:2408), G (SEQ ID NO:2409), H (SEQ ID NO:2410), and the WT N-ter sequence (SEQ ID NO:2411)). Figure 9B shows the sequences of all N-terminal sequences that were exchanged to create the Onconase variant constructs. [Figure 9C] Figure 9C depicts N-terminal variants of onconase and the results of comparative peptide yield studies. Figure 9C shows a comparison of peptide yields obtained after chemical cleavage for onconase constructs A through H and unmutated onconase. [Figure 10] Graph showing a comparison of MP18357 peptide quality produced with three different onconase variant constructs with modified N-termini, where SEQ ID NO: 1 is shown in the panel insert (upper right corner of each panel), compared to the HPLC profile of the original onconase construct. [Figure 11]

[0033] Figure 11A shows results from chemical cleavage optimization using the Onconase concatemer strategy. Figure 11A is a schematic of the Onconase concatemer strategy used. Different peptide sizes are generated when incomplete chemical cleavage of the concatemer occurs. Figure 11B shows RP-HPLC analysis demonstrating incomplete chemical digestion of the Onconase concatemer at 40°C. [Figure 12AB] Figure 12A shows graphs depicting the time course of peptide distribution percentage detected by RP-HPLC upon incubation with acetic acid. In Figure 12A, chemical cleavage was performed at 40°C. The Y-axis represents the percentage of each species (monopeptides, dipeptides, or tripeptides). In Figure 12B, chemical cleavage was performed at 60°C. The Y-axis represents the percentage of each species (monopeptides, dipeptides, or tripeptides). [Figure 12C]Figure 12C is a graph depicting the time course of peptide distribution percentage detected by RP-HPLC upon incubation with acetic acid. Chemical cleavage was carried out at 80°C. The Y-axis represents the percentage of each species (monopeptide, dipeptide, or tripeptide). [Figure 13] FIG. 13 is a graph showing a comparison of peptide release over time following chemical cleavage of concatemer onconase constructs at different temperatures. [Figure 14A] Figure 14A shows RP-HPLC analysis of a concatemer construct carrying three copies of the MP 18357 peptide. Figure 14A shows the RP-HPLC profiles (overlapping) obtained after solubilization with 20%, 30%, 40%, and 50% acetic acid using a protein concentration of 20 g / L at 60°C. [Figure 14B] Figure 14B shows RP-HPLC analysis of a concatemer construct carrying three copies of the MP 18357 peptide. Figure 14B shows the RP-HPLC profiles (overlapping) obtained after solubilization with 20%, 30%, 40%, and 50% acetic acid using a protein concentration of 50 g / L at 80°C. [Figure 14C] Figure 14C shows RP-HPLC analysis of a concatemer construct carrying three copies of the MP 18357 peptide. Figure 14C shows the RP-HPLC profiles (overlapping) obtained after solubilization with 20%, 30%, 40%, and 50% formic acid using a protein concentration of 20 g / L at 60°C. [Figure 14D] Figure 14D shows a graph depicting RP-HPLC analysis of a concatemer construct carrying three copies of the MP 18357 peptide. Figure 14D shows a comparison of RP-HPLC profiles (overlapping) obtained after solubilization with 20% formic acid or 20% acetic acid using a protein concentration of 50 g / L at 80°C. [Figure 15A] 15A is a schematic diagram of a peptide production protocol. Figure 15A is a schematic diagram of a chaotropic agent-free protocol for peptide production. [Figure 15B]15A-15C are schematic diagrams of peptide production protocols. Figure 15B is a schematic workflow protocol in which onconase solubilization requires the addition of a chaotropic agent. [Figure 16] 1 shows a comparison of peptide yields when peptide production is performed using a protocol without chaotropic agents and a protocol requiring the addition of chaotropic agents for onconase solubilization. Yields are expressed as mg / L of flask culture. [Figure 17] 1 is a graph showing the percentage disease control for peptides sprayed on tomato seedlings infected with gray mold (Botrytis cinerea). The peptides were sprayed at a concentration of 0.1 g / L or 0.3 g / L 24 hours after infection. [Figure 18AB] (FIG. 18A) Design of onconase fusion constructs with 1 to 6 copies of oligopeptide MP 18913. (FIG. 18B) Provides a table of total number of peptides, total number of N-terminal peptides, and total number of C-terminal peptides. [Figure 18C] FIG. 1 shows fusion protein expression using 10 μl / well and SDS-PAGE analysis showing, from left to right, ONC357_1x3, ONC357_2x3, ONC357_3x3, ONC357x3, ONC357x4, NEB P7717 molecular weight marker, ONC357x3, ONC357x4, ONC357x5, ONC357x6, and NEB P7717 molecular weight marker. [Figure 19] FIG. 1 shows HPLC analysis of peptide release following acid cleavage. [Figure 20A] FIG. 2 shows an amino acid sequence comparison between SEQ ID NO: 2412 and an onconase variant (SEQ ID NO: 2413) containing three lysine to alanine substitutions and a deletion of the six C-terminal histidines. [Figure 20B] FIG. 1 shows an alignment of several onconase variants (SEQ ID NOs: 2414 to 2421). [Figure 21A]Representative schematic diagrams of cells and batch cultures resulting from incorporation of inclusion bodies into E. coli cultures. Figure 21A shows a representative schematic diagram of an E. coli cell containing a DNA concatemer comprising a fusion carrier protein (also labeled with an asterisk), two amino acids used repeatedly as a linker ("XY"), and a peptide of interest ("PEP") positioned between the linkers. [Figure 21B] Representative schematic diagrams of cells and batch cultures resulting from incorporation of inclusion bodies into E. coli cultures. Figure 21B shows a representative bioreactor (left) in a batch scaling process for expressed peptide titer and E. coli cells (right) producing inclusion bodies (dashed gray line) as a result of activity of the incorporated DNA concatemers. DETAILED DESCRIPTION OF THE INVENTION

[0190] Provided herein are methods for producing oligopeptides, methods for releasing oligopeptides fused to insoluble carrier polypeptides that form inclusion bodies in cells, methods for purifying inclusion bodies containing the fusion proteins, and methods for producing fusion polypeptides. Also provided herein are insoluble carrier polypeptides (e.g., onconase polypeptides or TAF12 polypeptides) operably linked to two or more oligopeptides, and fusion polypeptides comprising modified insoluble carrier polypeptides (e.g., onconase polypeptides or TAF12 polypeptides), oligopeptides comprising active amino acid sequences.

[0191] The methods and polypeptides of the present disclosure are based, at least in part, on the applicant's discovery of a bioproduction method utilizing an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and a concatemeric protein construct comprising two or more oligopeptides that increases peptide yield and peptide quality over other bioproduction methods. The applicant's bioproduction method surprisingly improved the production of short, bioactive peptides, which are known in the art to be difficult and challenging to produce. In contrast to previous methods, the methods disclosed herein do not require the use of chaotropic agents or column-based purification steps, thus reducing the financial and technical burden of peptide bioproduction. The applicant has also developed mutant insoluble carrier polypeptides (e.g., an onconase polypeptide or a TAF12 polypeptide) that exhibit increased expression of inclusion bodies when expressed in host cells. Therefore, these mutant onconase polypeptides may be used as fusion partners to increase the bioproduction of a peptide of interest operably linked to the mutant onconase. Without wishing to be bound by theory, increased accumulation of the peptide of interest in inclusion bodies allows for increased levels of the peptide to be purified from the host cells.

[0192] As used herein, an "amino acid" or "amino acid residue" refers to any naturally occurring amino acid, any non-naturally occurring amino acid, any modified amino acid, including a derivatized amino acid, or any amino acid mimetic known in the art. Amino acids may be referred to by both their common three-letter abbreviations and their one-letter abbreviations.

[0193] As used herein, the term "peptide" refers to any peptide structure comprising or consisting of two or more amino acids, including chemical modifications and derivatives of amino acids. In some embodiments, peptides are short peptides (e.g., 4 to 50 amino acids in length). In some embodiments, peptides are oligopeptides that are released upon cleavage of the polypeptide.

[0194] As used herein, the term "purified" molecule refers to a biological or synthetic molecule that has been removed from its natural environment, isolated or separated, and free from other components with which it is naturally associated.

[0195] The term "isolated," in reference to molecules, including nucleic acids, constructs, vectors, etc., may refer to a molecule that is not found in nature and / or exists in a context in which it is not found in nature. The term "isolated" may also refer to a molecule that has been isolated from a more complex solution or source or that has undergone at least one step toward being concentrated or enriched. However, the term "isolated" is not intended to limit a molecule to a particular location or condition in any way. For example, an isolated nucleic acid molecule includes a nucleic acid molecule that has been introduced into the genome of a cell at a location not naturally found in the cell, or that resides in the progeny of a cell into which the nucleic acid molecule has been introduced at a location not naturally found in the genome of the cell.

[0196] The term "sequence identity" refers to the degree of similarity between two nucleic acid sequences or two amino acid sequences, expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is often measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

[0197] As used herein, a "concatemer polypeptide" or "concatemer" refers to a polypeptide that contains multiple copies of a given unit (e.g., oligopeptide) as a tandem repeat. In some embodiments, a concatemer polypeptide is a homoconcatemer (each oligopeptide unit is the same). In some embodiments, a concatemer polypeptide is a heteroconcatemer (there is more than one different oligopeptide). In some embodiments, a concatemer polypeptide contains linkers between units.

[0198] As used herein, "active amino acid sequence," "bioactive amino acid sequence," and "active sequence" refer to an amino acid sequence that can induce a biological effect in a living cell and / or organism exposed to the amino acid sequence. In some embodiments, an active amino acid sequence has antimicrobial activity. In some embodiments, an active amino acid sequence confers a benefit to a plant.

[0199] As used herein, an "onconase variant" is an onconase polypeptide that contains one or more amino acid insertions, deletions, or substitutions compared to the amino acid sequence set forth in SEQ ID NO:1.

[0200] I. Methods for Producing Oligopeptides Some aspects of the present disclosure provide methods for producing oligopeptides. In some embodiments, the methods for producing oligopeptides include expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to two or more oligopeptides by peptide bonds, and releasing the two or more oligopeptides from the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by sequence-specific chemical cleavage of the peptide bonds.

[0201] Fusion proteins In some embodiments, a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is operably linked to two or more oligopeptides. In some embodiments, a method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and two or more polypeptides. For example, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, or twenty or more oligopeptides. In some embodiments, a fusion polypeptide comprises two or more oligopeptides having the same amino acid sequence. In some embodiments, a fusion polypeptide comprises two or more oligopeptides having different amino acid sequences. In some embodiments, a fusion polypeptide comprises two or more oligopeptides having the same amino acid sequence and two or more oligopeptides comprising different amino acid sequences.

[0202] In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and two or more oligopeptides operably linked to the N-terminus and / or C-terminus of the onconase protein. For example, a fusion polypeptide can comprise two or more oligopeptides operably linked to each of the N-terminus and C-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, a fusion polypeptide comprises a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more oligopeptides operably linked to the N-terminus and / or C-terminus of the onconase protein. In some embodiments, the number of oligopeptides operably linked to each end of the onconase protein can be the same or different.

[0203] In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) with no oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and two or more oligopeptides operably linked to the C-terminus. In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) with no oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and four, five, or six oligopeptides operably linked to the C-terminus. In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and two or more oligopeptides operably linked to the N-terminus and three oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and one, two, or three oligopeptides operably linked to the N-terminus and three oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, one or more of the oligopeptides operably linked to the N-terminus of onconase may have the same amino acid sequence as one or more of the oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, one or more of the oligopeptides operably linked to the N-terminus of onconase have a different amino acid sequence than one or more of the oligopeptides operably linked to the C-terminus of onconase.In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and two or more oligopeptides linked to the C-terminus of onconase. In some embodiments, the two or more oligopeptides linked to the C-terminus of onconase have the same amino acid sequence. In some embodiments, the two or more oligopeptides linked to the C-terminus of onconase have different amino acid sequences. In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and two or more oligopeptides linked to the N-terminus of onconase. In some embodiments, the two or more oligopeptides linked to the N-terminus of onconase have the same amino acid sequence. In some embodiments, the two or more oligopeptides linked to the N-terminus of onconase have different amino acid sequences. In some embodiments, the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is separated from the two or more oligopeptides by a peptide bond. In some embodiments, the two or more oligopeptides are themselves separated by a peptide bond. In some embodiments, the peptide bond comprises a cleavable bond.

[0204] The cleavable bond may be cleaved by an enzymatic or chemical agent. In some embodiments, the cleavable bond is a peptide bond. Cleavage of the peptide bond may separate the oligopeptide from the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and / or from each other. Any cleavable peptide bond that is cleavable by any method or agent in the art may be used with the present methods. Proteolytic enzymes and their respective cleavage site specificities are well known in the art. Examples of enzymes useful for cleaving peptide linkers include, but are not limited to, Arg-C proteinase, Asp-N endopeptidase, chymotrypsin, clostripain, enterokinase, factor Xa, glutamyl endopeptidase, granzyme B, Achromobacter proteinase I, pepsin, proline endopeptidase, proteinase K, Staphylococcus aureus peptidase I, thermolysin, thrombin, trypsin, TEV protease, HRV3C, and members of the caspase family of proteolytic enzymes (e.g., caspases 1-10). Chemical cleavage agents are also well known in the art and can be used to cleave polypeptides at peptide bonds between specific pairs of amino acids. Examples of chemical cleavage reagents include cyanogen bromide (which cleaves after methionine residues, leaving a C-terminal homoserine lactone in place of the methionine), N-chlorosuccinimide, iodobenzoic acid, or BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole] (each of which cleaves tryptophan residues, leaving a C-terminal tryptophan (along with a lactone)), dilute acid (which cleaves aspartyl-prolyl bonds, leaving a C-terminal aspartic acid and an N-terminal proline), and hydroxylamine (which cleaves at asparagine-glycine bonds at pH 9.0, leaving a C-terminal asparagine and an N-terminal glycine).Additional chemical cleaving agents are described in Gavit, P. and Better, M., J. Biotechnol., 79: 127-136 (2000); Szoka et al., DNA, 5(1): 11-20 (1986); and Walker, J.M., The Proteomics Protocols Handbook, 2005, Humana Press, Totowa, NJ. In some embodiments, the peptide cleavable bond between peptides is cleavable with the same reagent as the peptide cleavable bond between the peptide and onconase. In some embodiments, the peptide cleavable bond between peptides is cleavable with a different reagent from the peptide cleavable bond between the peptide and onconase.

[0205] In some embodiments, the cleavage is at a specific amino acid sequence. In some embodiments, (i) the peptide bond contains methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond contains tryptophan, and the sequence-specific chemical cleavage uses BNPS skatole; (iii) the peptide bond contains aspartic acid-proline (Asp-Pro) bond, and the sequence-specific chemical cleavage uses formic acid, acetic acid, or any dilute acid solution; (iv) the peptide bond contains asparagine-glycine bond, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond contains cysteine, and the sequence-specific chemical cleavage uses NTCB. In some embodiments, two or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by a peptide bond containing an Asp-Pro bond, and the sequence-specific chemical cleavage uses formic acid or acetic acid. In some embodiments, the peptides are linked to each other by a peptide bond containing an Asp-Pro bond, and the sequence-specific chemical cleavage uses formic acid or acetic acid.

[0206] The two or more oligopeptides can correspond to any desired amino acid sequence. In some examples, the oligopeptide can be a small peptide (e.g., 4-25 amino acids in length). For example, the oligopeptide can be at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. The oligopeptide can alternatively be a longer peptide (e.g., up to 50 amino acids in length). The oligopeptide can be less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some examples, the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0207] In some embodiments, the one or more oligopeptides comprise the amino acid sequence of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:14.

[0208] In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) linked to two or more miPEPs. miPEPs are short peptides (7-44 amino acids in length) defined by one or several short open reading frames located in the pri-microRNA sequence of a specific miRNA member. Without wishing to be bound by theory, miPEPs target the pri-microRNA of their encoding miRNA to up- or down-regulate transcription. miPEPs may target miRNAs involved in various processes, such as miRNAs involved in immunity and susceptibility to pathogens (e.g., microorganisms), organogenesis, stress response, embryonic development, etc. In some embodiments, each of the two or more oligopeptides comprises a miPEP sequence that regulates a miRNA. In some embodiments, the two or more oligopeptides comprise a miPEP sequence that regulates one or more miRNA families. In some embodiments, each of the two or more oligopeptides comprises a miPEP sequence that regulates one or more members of a specific miRNA family. In some embodiments, each of the two or more oligopeptides comprises a miPEP sequence that regulates a plant miRNA, a fungal miRNA, or a metazoan miRNA. In some embodiments, each of the two or more oligopeptides comprises a miPEP sequence that regulates a plant miRNA. Exemplary plant microRNA families that can be regulated by miPEPs include, but are not limited to, the plant miRNA families miR156, miR159 / 319, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR390, miR393, miR394, miR295, miR396, miR397, miR398, miR408, miR403, miR437, miR444, and miR445. In some embodiments, each of the two or more oligopeptides comprises a miPEP sequence that has microbe inhibitory activity.

[0209] In some embodiments, the oligopeptide comprises any other peptide of interest. In some embodiments, the oligopeptide comprises a polypeptide that is toxic to host cells. In some embodiments, the oligopeptide comprises a therapeutic polypeptide, a toxin, a cytokine, a peptide hormone, a clotting factor, an immunogenic peptide, an allergen, an antimicrobial peptide, a ligand-binding domain, or any combination thereof. In some embodiments, an immunogenic peptide is a peptide that can induce an immune response against a polypeptide that includes the peptide. In some embodiments, one or more oligopeptides comprise a peptide fragment of a larger polypeptide. For example, one or more oligopeptides can comprise a peptide fragment of a receptor, an enzyme, an adhesion molecule, or a structural protein.

[0210] In some embodiments, the oligopeptide comprises an active amino acid sequence and a tag at the N-terminus and / or C-terminus. In some embodiments, the N-terminal and / or C-terminal residues may correspond to a cleavage tag that is generated after release of the oligopeptide. For example, the residue may be part of a peptide bond that is cleaved by a chemical reaction (e.g., acid cleavage) or an enzymatic reaction. In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the active amino acid sequence has antibacterial activity.

[0211] The fusion polypeptide may also include a linker sequence between two or more oligopeptides and / or between the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the oligopeptide. The linker sequence may function as a spacer peptide to separate the two or more oligopeptides and / or the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the oligopeptide. Any number or linker sequence known in the art may be used. In some embodiments, the linker is about 1 to 50 amino acids in length. In some embodiments, the linker is about 4 to 8 amino acids in length. In some embodiments, the linker sequence is a flexible amino acid sequence. In some embodiments, the flexible linker comprises glycine, serine, or threonine residues, or any combination thereof. In some embodiments, the flexible linker sequence comprises a (GGGGS)n (SEQ ID NO: 2402) or (G)n sequence, where n is an integer. In some embodiments, the flexible linker comprises the amino acid sequence GGG, GSGS (SEQ ID NO: 30), GSGSGGT (SEQ ID NO: 31), GGSGTG (SEQ ID NO: 27), or GTGSGTG (SEQ ID NO: 32). In some embodiments, the linker sequence is a rigid linker sequence. In some embodiments, the rigid linker sequence comprises an (EAAAK)n (SEQ ID NO: 33) or (XP)n sequence, where n is an integer and X is any amino acid. In some embodiments, the linker sequence comprises a flexible linker sequence and a rigid linker sequence. The linker sequence may also provide a cleavable peptide bond by incorporation of a cleavable linker sequence comprising an amino acid sequence that directs sequence-specific cleavage of the fusion polypeptide. In some embodiments, the linker sequence may comprise a sequence for protease or chemical cleavage. In some embodiments, the fusion polypeptide comprises a linker sequence comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion polypeptide comprises a linker sequence comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion polypeptide comprises a linker sequence comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the linker comprises an Asp-Pro sequence.

[0212] In some embodiments, the fusion polypeptide comprises a linker sequence separating two or more oligopeptides and / or a linker sequence separating an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from two or more oligopeptides. In some embodiments, the linker sequence separating two or more oligopeptides has the same sequence as the linker separating the onconase sequence from the oligopeptide. In some embodiments, the linker sequence between the oligopeptides is different from the linker sequence separating the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from two or more oligopeptides. In some embodiments, the linker sequence provides a cleavable peptide bond. In some embodiments, the linker sequence comprises an amino acid sequence for sequence-specific cleavage of the linker. In some embodiments, the sequence-specific cleavage is acid cleavage. In some embodiments, the fusion polypeptide comprises a linker sequence separating two or more oligopeptides and / or a linker sequence separating an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from two or more oligopeptides comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion polypeptide comprises a linker sequence comprising an Asp-Pro linker separating two or more oligopeptides and / or separating an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from two or more oligopeptides.

[0213] In some embodiments, the fusion polypeptide further comprises one or more amino acid sequence tags. In some embodiments, the amino acid sequence tag does not affect the solubility of the onconase fusion protein. In some embodiments, the amino acid sequence tag has a neutral pI. In some embodiments, the one or more amino acid sequence tags are purification tag peptides. Such tag peptides may include, but are not limited to, glutathione-S-transferase (GST), polyhistidine, maltose-binding protein (MBP), avidin, biotin, streptavidin, histidine (His) tag (e.g., His-6X tag), V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, thioredoxin (Trx) tag, and ligands for cellular receptors (e.g., insulin receptor ligands). In some embodiments, the one or more amino acid sequence tags are reporter tag peptide sequences. Examples of reporter tag peptide sequences include, but are not limited to, horseradish peroxidase (HRP), chromamphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, and fluorescent proteins (e.g., GFP, CFP, YFP, BFP, etc.).

[0214] In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus and / or C-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) or two or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus and / or C-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, one or more tag peptides are operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, one or more tag peptides are operably linked to the N-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus and / or C-terminus of two or more oligopeptides. In some embodiments, one or more tag peptides are operably linked to the N-terminus of two or more oligopeptides. In some embodiments, one or more tag peptides are operably linked to the C-terminus of two or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and to the N-terminus of two or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus of onconase and the C-terminus of one or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of one oligopeptide and the N-terminus of another oligopeptide. In some embodiments, the amino acid sequence tag operably linked to the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is the same as the amino acid sequence tag operably linked to two or more oligopeptides.In some embodiments, the amino acid sequence tag operably linked to the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is different from the amino acid sequence tags operably linked to the two or more oligopeptides.

[0215] Onconase is an RNAse that exhibits a pH-dependent change in solubility. The solubility of onconase is determined by its isoelectric point (pI). The pI of a protein is the pH at which the protein carries no net charge. In acidic conditions, where the pH is lower than the pI, onconase is positively charged and soluble. In neutral conditions, where the pH is approximately the pI, onconase is neutrally charged and forms insoluble aggregates. These insoluble aggregates can be removed from solution by selective precipitation. The pI corresponds to the average acid dissociation constant, or pKa, of the amino acids in a particular protein. Thus, a protein's pI is higher if it contains more Arg (pKa = 12.48), Lys (pKa = 10.79), Tyr (pKa = 10.07), Cys (pKa = 8.35), His (pKa = 6.04), Asp (pKa = 3.86), and Glu (pKa = 4.25) residues, and lower if it contains more nonpolar amino acid residues. Onconase is a highly cationic protein, with a pI greater than 9.5. In some embodiments, onconase has a pI of approximately 7.5. In some embodiments, onconase has a neutral pI. Furthermore, in some embodiments, onconase lacks any aspartic acid-proline and asparagine-glycine sequences, which makes it resistant to common chemical cleavage strategies, such as acid cleavage.

[0216] In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising the amino acid sequence of SEQ ID NO: 1 operably linked by peptide bonds to two or more oligopeptides. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide is a truncated onconase polypeptide.

[0217] In some embodiments, the fusion polypeptide comprises a mutant onconase polypeptide operably linked to two or more oligopeptides by peptide bonds. The mutant onconase may be an onconase having a mutation that reduces susceptibility to proteolytic cleavage, increases yield, promotes aggregation, or promotes solubility. For example, M23L onconase (M23L-ONC) disclosed by Pane et al. (Pane, K et al. (2016) PLoS One. 1 l(l):e0146552, incorporated herein by reference) does not contain any internal methionine residues and is less susceptible to chemical cleavage strategies. The modification may be one or more of an amino acid substitution, deletion, or insertion. Other amino acid substitutions, insertions, or deletions that increase the stability, yield, or aggregation of the onconase protein may also be introduced.

[0218] In some embodiments, the fusion polypeptide comprises a mutant onconase containing one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are conservative amino acid substitutions. A conservative amino acid substitution refers to the substitution of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. In some embodiments, the one or more amino acid substitutions are non-conservative amino acid substitutions. A non-conservative amino acid substitution refers to the substitution of amino acids with different properties (e.g., an uncharged residue replaced with a charged residue). In some embodiments, the fusion polypeptide comprises an onconase polypeptide that comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO: 1. The one or more insertions or deletions can be of any amino acid length.

[0219] In some embodiments, a fusion protein comprises an onconase polypeptide with one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide with one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0220] In some embodiments, a fusion protein comprises an onconase polypeptide having one or more amino acid substitutions that increase the yield or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions that increase the yield or promote the formation of inclusion bodies comprise one or more substitutions of cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, the onconase comprises one or more amino acid substitutions, insertions, or deletions that decrease solubility. In some embodiments, the one or more amino acid substitutions result in a mutant onconase polypeptide having a reduced number of cationic or polar amino acids compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions that increase the yield or promote the formation of inclusion bodies result in a mutant onconase having a reduced number of Lys, Arg, Cys, His, Asp, Glu, and / or Tyr compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide having one or more amino acid substitutions that result in an onconase protein with an altered pI compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide having one or more deletions or insertions that increase the yield of or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more insertions or deletions that increase the yield of or promote the formation of inclusion bodies result in a mutant onconase that has a reduced number of cationic or polar amino acids compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more insertions or deletions that increase the yield of or promote the formation of inclusion bodies result in a mutant onconase that has a reduced number of Lys, Arg, Cys, His, Asp, Glu, and / or Tyr compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1.In some embodiments, a fusion protein comprises an onconase polypeptide having one or more deletions or insertions that increase the yield of or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide having one or more deletions or insertions that result in an onconase protein with an altered pI compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In any of the above embodiments, the onconase polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 1.

[0221] In some embodiments, a fusion protein comprises an onconase polypeptide having one or more N-terminal amino acid substitutions relative to SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide having 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 N-terminal amino acid substitutions relative to SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide having 11 N-terminal amino acid substitutions relative to SEQ ID NO: 1. In some embodiments, the amino acid substitutions are within the first 50 N-terminal amino acids, within the first 40 N-terminal amino acids, within the first 30 N-terminal amino acids, within the first 20 N-terminal amino acids, or within the first 11 N-terminal amino acids. In some embodiments, the amino acid substitutions are within the first 15 N-terminal amino acids, the first 14 N-terminal amino acids, the first 13 N-terminal amino acids, the first 12 N-terminal amino acids, the first 11 N-terminal amino acids, the first 10 N-terminal amino acids, the first 9 N-terminal amino acids, the first 8 N-terminal amino acids, the first 7 N-terminal amino acids, the first 6 N-terminal amino acids, the first 5 N-terminal amino acids, the first 4 N-terminal amino acids, the first 3 N-terminal amino acids, or the first 2 N-terminal amino acids. In some embodiments, one or more N-terminal amino acid substitutions are conservative amino acid substitutions. In some embodiments, one or more N-terminal amino acid substitutions are non-conservative amino acid substitutions. In some embodiments, a fusion protein comprises an onconase polypeptide having one or more N-terminal insertions or deletions compared to SEQ ID NO:1. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising the amino acid sequence of SEQ ID NO:1. In some embodiments, the fusion polypeptide comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1.In some embodiments, the fusion polypeptide comprises an onconase polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO:1.

[0222] In some embodiments, a fusion protein comprises an onconase polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-10 or 15-22, optionally with one, two, three, four, five, or all six C-terminal histidine residues deleted, as shown in Figure 20A. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:5.In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:9.In some embodiments, the fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18.In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the fusion polypeptide comprises an onconase polypeptide comprising at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 2-10 or 15-22, and optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20A.

[0223] In some embodiments, a fusion polypeptide comprises an onconase polypeptide having an altered N-terminus and / or C-terminus. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID NO: 1.

[0224] In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO: 23 operably linked by peptide bonds to two or more oligopeptides. In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 23. In some embodiments, the TAF12 polypeptide is a truncated TAF12 polypeptide.

[0225] In some embodiments, the fusion polypeptide comprises a mutant TAF12 polypeptide operably linked to two or more oligopeptides by peptide bonds. The mutant TAF12 may be a TAF12 having a mutation that reduces susceptibility to proteolytic cleavage, increases yield, promotes aggregation, or promotes solubility. The modification may be one or more of an amino acid substitution, deletion, or insertion. Other amino acid substitutions, insertions, or deletions that increase the stability, yield, or aggregation of the TAF12 protein may also be introduced.

[0226] In some embodiments, the fusion polypeptide comprises a mutant TAF12 comprising one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more amino acid substitutions are conservative amino acid substitutions. A conservative amino acid substitution refers to the substitution of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. In some embodiments, the one or more amino acid substitutions are non-conservative amino acid substitutions. A non-conservative amino acid substitution refers to the substitution of amino acids with different properties (e.g., an uncharged residue replaced with a charged residue). In some embodiments, the fusion protein comprises a TAF12 polypeptide that comprises one or more insertions or deletions relative to the amino acid sequence of SEQ ID NO: 23. The one or more insertions or deletions can be of any amino acid length.

[0227] In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions that decrease its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions that decrease its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23.

[0228] In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions that increase the yield or promote the formation of inclusion bodies compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more amino acid substitutions that increase the yield or promote the formation of inclusion bodies comprise one or more substitutions of cationic or polar amino acids for nonpolar or neutral amino acids. In some embodiments, the TAF12 comprises one or more amino acid substitutions, insertions, or deletions that reduce solubility. In some embodiments, the one or more amino acid substitutions result in a mutant TAF12 polypeptide having a reduced number of cationic or polar amino acids compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more amino acid substitutions that increase the yield or promote the formation of inclusion bodies result in a mutant TAF12 polypeptide having a reduced number of Lys, Arg, Cys, His, Asp, Glu, and / or Tyr compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions resulting in a TAF12 protein with an altered pI compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions that increase the yield of or promote the formation of inclusion bodies compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more insertions or deletions that increase the yield of or promote the formation of inclusion bodies result in a mutant TAF12 having a reduced number of cationic or polar amino acids compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more insertions or deletions that increase the yield of or promote the formation of inclusion bodies result in a mutant TAF12 having a reduced number of Lys, Arg, Cys, His, Asp, Glu, and / or Tyr compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23.In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions that increase the yield of or promote the formation of inclusion bodies compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions that result in a TAF12 protein with an altered pI compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In any of the above embodiments, the TAF12 polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 23.

[0229] In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more N-terminal amino acid substitutions relative to SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide having 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 N-terminal amino acid substitutions relative to SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide having 11 N-terminal amino acid substitutions relative to SEQ ID NO: 23. In some embodiments, the amino acid substitutions are within the first 50 N-terminal amino acids, within the first 40 N-terminal amino acids, within the first 30 N-terminal amino acids, within the first 20 N-terminal amino acids, or within the first 11 N-terminal amino acids. In some embodiments, the amino acid substitutions are within the first 15 N-terminal amino acids, the first 14 N-terminal amino acids, the first 13 N-terminal amino acids, the first 12 N-terminal amino acids, the first 11 N-terminal amino acids, the first 10 N-terminal amino acids, the first 9 N-terminal amino acids, the first 8 N-terminal amino acids, the first 7 N-terminal amino acids, the first 6 N-terminal amino acids, the first 5 N-terminal amino acids, the first 4 N-terminal amino acids, the first 3 N-terminal amino acids, or the first 2 N-terminal amino acids. In some embodiments, one or more N-terminal amino acid substitutions are conservative amino acid substitutions. In some embodiments, one or more N-terminal amino acid substitutions are non-conservative amino acid substitutions. In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more N-terminal insertions or deletions compared to SEQ ID NO:23. In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO:23. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23.In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO:23.

[0230] Peptide release In some embodiments, methods for producing oligopeptides include releasing two or more oligopeptides from an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by sequence-specific cleavage of peptide bonds. In some embodiments, the sequence-specific cleavage of peptide bonds is performed by acid cleavage. In some embodiments, the acid cleavage is performed by incubation with acetic acid or formic acid. In some embodiments, the incubation uses an elevated temperature, such as above room temperature.

[0231] In some embodiments, the oligopeptides are operably linked by peptide bonds and are released from one another when the oligopeptides are released from onconase, hi some embodiments, the oligopeptides are operably linked by different peptide bonds and are released from one another by sequence-specific chemical cleavage of the different peptide bonds after the oligopeptides are released from onconase.

[0232] In some embodiments, the step of releasing two or more oligopeptides from an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is carried out in the presence of a chaotropic agent. Chaotropic agents are molecules that can disrupt the hydrogen bond network between water molecules. It is theorized that the addition of chaotropic agents can reduce the structural order of proteins, thus promoting protein unfolding. Examples of chaotropic agents include, but are not limited to, guanidine hydrochloride (guanidinium hydrochloride, GdnHCl), sodium thiocyanate, n-butanol, ethanol, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, sodium dodecyl sulfate, and urea. A chaotropic agent may also be a detergent that disrupts non-covalent intermolecular bonds within proteins, causing amino acid chains to adopt substantially random conformations.

[0233] In some embodiments, the step of releasing two or more oligopeptides from an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is performed in the absence of a chaotropic agent. The addition of a chaotropic agent may not always be desirable in a protein purification protocol. For example, the addition of a chaotropic agent may require additional steps or conditions to be added to the protocol. These additional steps may increase the amount of time, reagents, and expense of the purification protocol, or may adversely affect protein yield or purity.

[0234] Fusion polypeptides can be expressed in any suitable cell. Fusion polypeptides can be expressed in bacterial cells, yeast cells, plant cells, or animal cells. In some embodiments, fusion polypeptides are expressed in unicellular organisms. In some embodiments, fusion polypeptides are expressed in microbial cells. Any cell or cell culture known to produce inclusion bodies is suitable for expressing fusion polypeptides. For example, fusion polypeptides can be expressed in strains of Escherichia coli and Vibrio natrigens. Suitable yeast cells include, but are not limited to, strains of Pichia pastoris. The bacterial strain can be a strain lacking Lon and ompT protease functions (e.g., a BL21 E. coli strain). In some embodiments, fusion polypeptides are expressed in bacteria in a bacterial cell culture. In some embodiments, the bacteria is an E. coli strain.

[0235] Expression of the peptide may be driven by any suitable promoter known in the art. The promoter may be a constitutive promoter or an inducible promoter. An inducible promoter is a regulated promoter that can be activated by an external stimulus (e.g., chemical, nutritional stress, or heat). For example, the lac promoter can be induced through the use of lactose or IPTG (isopropylthio-β-D-galactoside). Alternatively, the promoter may be a constitutive promoter, which directs expression of the fusion polypeptide in a particular organism, cell, or tissue. The strength of the promoter, whether inducible or constitutive, may be varied. For example, the promoter may be a high-expression promoter or a strong promoter, which results in overexpression of a given gene. In some embodiments, the fusion polypeptide is expressed by a strong promoter. In some embodiments, the promoter drives expression of the fusion protein in aggregates.

[0236] In some embodiments, the method further comprises measuring the yield of the released oligopeptide. Protein yield may be measured by any known method for measuring peptide yield. For example, peptide yield may be measured in terms of peptide mass obtained from 1 liter of cell culture, or g / L. In some embodiments, the oligopeptide yield is 10 mg per L of bacterial culture, 20 mg per L of bacterial culture, 30 mg per L of bacterial culture, 40 mg per L of bacterial culture, or 50 mg per L of bacterial culture. In some embodiments, the yield of oligopeptide is between 10 mg per L and 200 mg per L of bacterial culture, between 20 mg per L and 150 mg per L, between 30 mg per L and 120 mg per L of bacterial culture, between 40 mg per L and 120 mg per L of bacterial culture, between 50 mg per L and 120 g per L of bacterial culture, between 50 g per L and 100 g per L of bacterial culture, between 60 g per L and 90 g per L of bacterial culture, between 70 g per L and 110 g per L of bacterial culture, or between 40 g per L and 80 g per L of bacterial culture. In some embodiments, the yield of expressed fusion polypeptide is measured after harvesting the cells at stationary phase.

[0237] In some embodiments, the method may include expressing the fusion polypeptide in a cell culture in a bioreactor. Use of a bioreactor may result in higher yields of the released oligopeptide. Large-scale cell growth and fusion polypeptide expression may utilize a wide range of simple or complex carbohydrates, organic acids or alcohols, and saturated hydrocarbons such as methane. Fusion protein encoding gene expression may be regulated, repressed, or reduced by specific growth conditions, which may include the form and amount of any trace micronutrient, including nitrogen, phosphorus, sulfur, oxygen, carbon, or small inorganic ions. Furthermore, regulation may be achieved by the presence or absence of specific regulatory molecules added to the culture that are not typically considered nutrients or energy sources.

[0238] II. Methods for releasing oligopeptides from insoluble carrier polypeptides that form inclusion bodies Some aspects of the present disclosure provide methods for releasing oligopeptides fused to insoluble carrier polypeptides that form inclusion bodies in cells.

[0239] Inclusion bodies are intracellular amorphous precipitates containing aggregated proteins found in the cytoplasm or periplasm of cells. A target oligopeptide, typically soluble in cells and / or cell lysates, can be linked to an insoluble carrier polypeptide, which forms inclusion bodies that promote the formation of insoluble aggregates containing the oligopeptide. Once trapped in inclusion bodies, the oligopeptide becomes inaccessible to cellular proteases and can accumulate in large quantities. Examples of insoluble carrier polypeptides that form inclusion bodies include, but are not limited to, trpΔLE polypeptides (Derynck R et al., (1984)), ketosteroid isomerase polypeptides (Kuliopulos et al., (1994)), β-galactosidase polypeptides (Schellenberger V et al., (1993)), PagP polypeptides (Hwang PM et al., (2012)), EDDIE (Achmuller C et al., (2007)), the cleavable self-aggregating tag INTEIN-ELK16 (Zhao Q et al., (2016)), GFIL8 (Wang X. et al., (2015)), PaP3.30 polypeptides (Rao XC et al., (2004)), TAF12- HFD (Vidovic V et al., (2012)), and the F4 fragment of PurF (Lee JH et al., (2000)).Other representative examples of insoluble carrier polypeptides that form inclusion bodies include modified versions of E. coli maltose-binding protein (Betton and Hofhug, J. Biol. Chem. 271:8046-8052 (1996)), E. coli RNAse II polypeptides (Cobum and Mackie, J. Biol. Chem. 271:1048-1053 (1996)), E. coli alkaline phosphatase polypeptides (Derman and Beckwith, J. Bacteriol. 177:3764-3770 (1995); Georgiou et al., Appl. Env. Microbial. 52:1157-1161 (1986)), E. coli phospholipase A polypeptides (Dekker et al., Eur. J. Biochem. 232:214-219 (1996)), and the like. (1995)), Escherichia coli β-lactamase polypeptide (Rinas and Bailey, Appl. Env. Microbiol. 59:561-566 (1993); Georgiou et al., Appl. Env. Microbiol. 52: 1157-1161 (1986)), Salmonella typhimurium MalK protein (Schneider et al., Prot. Exp. Purif. 6: 10-14 (1995)), Clostridium thermocellum endoglucanase D polypeptide (Tokatlidis et al., FEBS Lett. 282:205-208 (1991)), Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein (Oeda et al., J. Bacteriol. 171:3568-3571 (1991)), (1989), human procathepsin B polypeptide (Kuhelj et al., Eur. J. Biochem. 229:533-539 (1995)), a porcine interferon-γ polypeptide (Vandenbroeck et al., Eur. J. Biochem. 215:481-486 (1993)), T5 DNA polymerase polypeptide (Chatterjee et al., Gene 97: 13-19 (1991)), and E. coli thioredoxin (Hoog et al., BioSci. Rep. 4:917-923 (1984)).In some embodiments, the insoluble carrier polypeptide is an onconase polypeptide or a TAF12 polypeptide.

[0240] In some embodiments, a method for releasing oligopeptides fused to an insoluble carrier polypeptide that forms inclusion bodies in cells includes expressing a fusion polypeptide comprising an oligopeptide operably linked to an insoluble carrier polypeptide that forms inclusion bodies in cells, and releasing two or more oligopeptides from the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by sequence-specific chemical cleavage of peptide bonds.

[0241] In some embodiments, the method includes purifying inclusion bodies. The inclusion bodies may be purified by lysing the cells and extracting the inclusion bodies. The cells may be lysed using any number of means, including mechanical and / or chemical lysis. Any mechanical method of purifying inclusion bodies from cell lysates known in the art may be used, including centrifugation, filtration, sonication, French press, shearing, and any combination thereof.

[0242] Inclusion bodies may be isolated by solubilizing the aggregates using micelles or reverse micelles, for example, as described in Vinogradov et al. (2003) Anal Biochem. 15; 320(2):234-8. In some embodiments, purifying the inclusion bodies comprises centrifuging the inclusion bodies. In some embodiments, centrifuging the inclusion bodies separates the inclusion bodies from the cell lysate. In some embodiments, the inclusion bodies are solubilized after purification in a solution containing a chemical cleavage agent. In some embodiments, the inclusion bodies are solubilized after purification in a solution without a chaotropic agent. In some embodiments, the method comprises purifying inclusion bodies formed by a fusion protein comprising onconase fused to two or more oligopeptides. In some embodiments, the fusion protein is expressed in bacteria or yeast. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the oligopeptide is 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the onconase is a mutant onconase.

[0243] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in cells, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, further comprising incubating the inclusion bodies with acetic acid. In some embodiments, the inclusion bodies are incubated with acetic acid at an elevated temperature. In some embodiments, the temperature is greater than 50°C, greater than 60°C, greater than 70°C, greater than 80°C, or greater than 90°C. In some embodiments, the temperature is less than 100°C, less than 95°C, less than 90°C, or less than 85°C. In some embodiments, the temperature is between 50°C and 60°C, between 60°C and 70°C, between 70°C and 80°C, between 80°C and 90°C, between 50°C and 100°C, or between 90°C and 100°C. In some embodiments, the method includes expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to two or more polypeptides by peptide bonds. In some embodiments, the peptide bond can be subjected to sequence-specific chemical cleavage. In some embodiments, two or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage uses formic acid or acetic acid.

[0244] In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50°C for at least 1 hour. The acetic acid concentration may be at least 2 weight percent, at least 3 weight percent, at least 4 weight percent, or at least 5 weight percent. In some embodiments, the acetic acid concentration is less than 50 weight percent, less than 45 weight percent, less than 40 weight percent, less than 35 weight percent, or less than 30 weight percent. In some embodiments, the acetic acid concentration is less than 50 weight percent, less than 45 weight percent, less than 40 weight percent, less than 35 weight percent, or less than 30 weight percent. In some embodiments, the acetic acid concentration is between 10 and 20 weight percent, between 20 and 30 weight percent, between 30 and 40 weight percent, or between 40 and 50 weight percent. In some embodiments, the inclusion bodies are incubated in acetic acid for 1 to 24 hours. In some embodiments, the inclusion bodies are incubated in acetic acid for 2 to 4 hours, 4 to 6 hours, 6 to 8 hours, 8 to 10 hours, 10 to 14 hours, 14 to 18 hours, 18 to 20 hours, or 20 to 24 hours. In some embodiments, the inclusion bodies are incubated in acetic acid for about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, or about 24 hours. In some embodiments, the inclusion bodies are incubated in acetic acid for up to 20 hours. In some embodiments, the method includes expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to two or more polypeptides by peptide bonds. In some embodiments, the peptide bonds are capable of sequence-specific chemical cleavage. In some embodiments, two or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage uses formic acid or acetic acid.

[0245] In some embodiments, a method for purifying inclusion bodies comprises expressing a fusion protein in cells, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, further comprising releasing two or more oligopeptides from an insoluble carrier polypeptide that forms inclusion bodies in the cells by sequence-specific chemical cleavage. In some embodiments, releasing two or more oligopeptides from an insoluble carrier polypeptide that forms inclusion bodies in the cells is carried out using an acid. In some embodiments, the acid is acetic acid or formic acid. In some embodiments, releasing two or more oligopeptides from an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by sequence-specific acetic acid cleavage of peptide bonds. In some embodiments, cleavage of the fusion polypeptide with acetic acid releases the insoluble carrier polypeptide that forms inclusion bodies in the cells and / or releases the two or more oligopeptides from each other from the two or more polypeptides. In some embodiments, releasing two or more oligopeptides from an insoluble carrier polypeptide that forms inclusion bodies in the cells occurs after solubilization of the inclusion bodies. In some embodiments, releasing two or more oligopeptides from an insoluble carrier polypeptide that forms inclusion bodies in cells occurs during incubation of the inclusion bodies in acetic acid. In some embodiments, release of the peptides is incomplete. For example, in some embodiments, a dipeptide or tripeptide remains after incubation with acetic acid or formic acid.

[0246] In some embodiments, the method further comprises separating the released oligopeptide from the insoluble carrier polypeptide that forms inclusion bodies in the cells. After the release step, the oligopeptide can be separated and / or isolated from the insoluble carrier polypeptide that forms the inclusion bodies based on the differential solubility of the components. Parameters such as pH, salt concentration, and temperature can be adjusted to facilitate separation of the oligopeptide from the insoluble carrier polypeptide that forms the inclusion bodies. The released oligopeptide can be further purified using any purification technique known in the art, such as, for example, ion exchange, gel purification techniques, differential centrifugation, and column chromatography.

[0247] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in cells, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the method includes expressing the fusion polypeptide under conditions that promote the formation of inclusion bodies. In some embodiments, the step of expressing the fusion polypeptide is carried out at neutral pH. In some embodiments, expressing the fusion polypeptide at neutral pH results in the formation of inclusion bodies. In some embodiments, expressing the fusion polypeptide is carried out at elevated temperature. In some embodiments, expressing the fusion polypeptide at elevated temperature results in the formation of inclusion bodies. In some embodiments, expressing the fusion polypeptide at high levels results in the formation of inclusion bodies. In some embodiments, expressing the fusion polypeptide is carried out using cells that are prone to forming inclusion bodies. In some embodiments, expressing the fusion polypeptide in cells that are prone to forming inclusion bodies results in the formation of inclusion bodies.

[0248] In some embodiments, provided herein are methods for purifying inclusion bodies comprising a fusion protein, wherein the fusion protein comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to two or more oligopeptides. In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to two or more polypeptides by peptide bonds. In some embodiments, the peptide bonds are amenable to sequence-specific chemical cleavage. In some embodiments, the two or more oligopeptides are operably linked to the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by peptide bonds comprising an Asp-Pro bond, and the sequence-specific chemical cleavage uses formic acid or acetic acid.

[0249] In some embodiments, a method for purifying inclusion bodies containing a fusion protein comprises expressing a fusion polypeptide comprising two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more polypeptides, eight or more polypeptides, ten or more polypeptides, fifteen or more polypeptides, or twenty or more oligopeptides operably linked to an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and two or more oligopeptides operably linked to the N-terminus or C-terminus of the onconase protein. In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide), wherein two or more oligopeptides are linked to the C-terminus of onconase. In some embodiments, the two or more oligopeptides linked to the C-terminus of onconase have the same amino acid sequence. In some embodiments, the two or more oligopeptides linked to the C-terminus of onconase have different amino acid sequences. In some embodiments, the method includes expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide), wherein two or more oligopeptides are linked to the N-terminus of onconase. In some embodiments, the two or more oligopeptides linked to the N-terminus of onconase have the same amino acid sequence. In some embodiments, the two or more oligopeptides linked to the N-terminus of onconase have different amino acid sequences. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the onconase is an onconase variant. In some embodiments, the onconase variant comprises one or more amino acid substitutions, insertions, or deletions in the 15 N-terminal amino acids.

[0250] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in cells, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, e.g., at a temperature of at least 50°C for at least 1 hour, wherein the fusion polypeptide comprises two or more oligopeptides that are less than 25 amino acids in length. In some embodiments, the two or more oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the two or more oligopeptides are up to 50 amino acids in length. In some embodiments, the two or more oligopeptides are less than 50, 45, 40, 35, 30, 25, or 20 amino acids in length. In some embodiments, the two or more oligopeptides are between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50° C. for at least 1 hour.

[0251] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in cells, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) linked to two or more miPEPs. In some embodiments, the two or more oligopeptides comprise miPEP sequences that regulate miRNAs. In some embodiments, the two or more oligopeptides comprise miPEP sequences that regulate two or more miRNA families. In some embodiments, the two or more oligopeptides comprise miPEP sequences that regulate plant miRNAs. In some embodiments, the two or more oligopeptides comprise a miPEP sequence that regulates a plant miRNA selected from the group consisting of the miR156, miR159 / 319, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR390, miR393, miR394, miR295, miR396, miR397, miR398, miR408, miR403, miR437, miR444, and miR445 families. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that has microbe inhibitory activity. In some embodiments, the onconase is a mutant onconase. In some embodiments, the two or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the fusion polypeptide is expressed in yeast or bacterial cells. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50° C. for at least 1 hour.

[0252] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in cells, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the fusion polypeptide further comprises a linker sequence between two or more oligopeptides and / or between the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the oligopeptide. In some embodiments, the linker sequence is cleavable by sequence-specific chemical cleavage. In some embodiments, a linker sequence separates two or more oligopeptides and / or a linker sequence separates the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the two or more oligopeptides. In some embodiments, the linker sequence separating the two or more oligopeptides has the same or a different sequence as the linker sequence separating the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the two or more oligopeptides. In some embodiments, the method comprises expressing a fusion polypeptide comprising a linker sequence comprising the amino acid sequence of SEQ ID NO: 11, which separates two or more oligopeptides and / or separates an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the two or more oligopeptides. In some embodiments, the method comprises expressing a fusion polypeptide comprising a linker sequence comprising an Asp-Pro linker, which separates two or more oligopeptides and / or separates an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the two or more oligopeptides. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50°C for at least 1 hour.

[0253] In some embodiments, a method for purifying inclusion bodies comprises expressing a fusion protein in a cell, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the fusion polypeptide comprises an onconase polypeptide comprising the amino acid sequence of SEQ ID NO: 1 operably linked by peptide bonds to two or more oligopeptides. In some embodiments, the method comprises expressing a fusion polypeptide comprising an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the method comprises expressing a fusion polypeptide comprising an onconase polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 1. In some embodiments, the method includes expressing a fusion polypeptide comprising a mutant onconase comprising one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method includes expressing a fusion polypeptide comprising an onconase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO: 1. The one or more insertions or deletions can be of any amino acid length. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50°C for at least 1 hour.

[0254] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in a cell, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the fusion polypeptide comprises an onconase polypeptide having one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method comprises expressing a fusion polypeptide comprising an onconase polypeptide having one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50°C for at least 1 hour.

[0255] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in cells, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the fusion polypeptide includes an onconase polypeptide having a modified N-terminus and / or C-terminus. In some embodiments, the method includes expressing a fusion polypeptide including an onconase polypeptide including one or more amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO:1. In some embodiments, the method includes expressing a fusion polypeptide including an onconase polypeptide including 11 amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO:1. In some embodiments, the method includes expressing a fusion polypeptide including an onconase polypeptide including 11 amino acid substitutions at the N-terminus compared to SEQ ID NO:1. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50° C. for at least 1 hour.

[0256] In some embodiments, a method for purifying inclusion bodies comprises expressing a fusion protein in a cell, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the fusion polypeptide comprises a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO: 23 operably linked by peptide bonds to two or more oligopeptides. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a mutant TAF12 comprising one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide comprising one or more insertions or deletions relative to the amino acid sequence of SEQ ID NO: 23. The one or more insertions or deletions may be of any amino acid length. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50°C for at least 1 hour.

[0257] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in a cell, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the fusion polypeptide comprises a TAF12 polypeptide having one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide having one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50°C for at least 1 hour.

[0258] In some embodiments, a method for purifying inclusion bodies includes expressing a fusion protein in cells, purifying the inclusion bodies, and incubating the inclusion bodies with acetic acid, wherein the fusion polypeptide comprises a TAF12 polypeptide having a modified N-terminus and / or C-terminus. In some embodiments, the method includes expressing a fusion polypeptide comprising a TAF12 polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, the method includes expressing a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, the method includes expressing a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus relative to SEQ ID NO:23. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50° C. for at least 1 hour.

[0259] III. Methods for Purifying Inclusion Bodies Containing Fusion Proteins Some aspects of the present disclosure provide methods for purifying inclusion bodies comprising a fusion protein. In some embodiments, the methods include expressing a fusion protein comprising an oligopeptide (e.g., onconase) operably linked to an inclusion body-forming protein, lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, washing the pellet in a detergent buffer, washing the pellet in a salt buffer, and washing the pellet in water. In some embodiments, the methods include purifying inclusion bodies formed by a fusion protein comprising onconase fused to two or more oligopeptides. In some embodiments, the fusion protein is expressed in bacteria or yeast. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acids in length. In some embodiments, the onconase is an onconase variant.

[0260] In some embodiments, a method for purifying inclusion bodies containing a fusion polypeptide includes lysing cells to form a lysate. Cell lysis can be performed by any method known to those of skill in the art. In some embodiments, cell lysis is performed by enzymatic lysis. Methods for enzymatic lysis include, but are not limited to, enzymatic lysis using lytic enzymes such as lysozyme, lysostaphin, mutanolysin, or any other enzyme that disrupts cell walls. In some embodiments, cell lysis is performed by mechanical methods. Methods for mechanical lysis include, but are not limited to, physical shearing, such as with glass beads, sonication, French press, ultrasound, incubation with hypotonic solutions, or high pressure. In some embodiments, the cell lysate is a yeast or bacterial cell lysate. In some embodiments, the fusion protein comprises onconase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the onconase is a mutant onconase.

[0261] In some embodiments, the method for purifying inclusion bodies containing a fusion polypeptide includes centrifuging a cell lysate to form a pellet. In some embodiments, the centrifugation is performed at a speed of about 500 x g, about 1,000 x g, about 5,000 x g, about 10,000 x g, about 15,000 x g, about 20,000 x g, or about 25,000 x g. In some embodiments, the centrifugation is performed at a speed between 500 x g and 1,000 x g, 1,000 x g and 5,000 x g, 5,000 x g and 10,000 x g, 10,000 x g and 15,000 x g, 15,000 x g and 20,000 x g, or 20,000 x g and 25,000 x g. In some embodiments, the centrifugation is performed at a speed of about 10,000 x g. In some embodiments, centrifugation is performed at a temperature of about 4°C to 10°C. In some embodiments, centrifugation is performed at room temperature. In some embodiments, centrifugation is performed for about 5 to 60 minutes. In some embodiments, centrifugation is performed for about 5 to 10 minutes, about 10 to 15 minutes, about 15 to 30 minutes, about 30 to 45 minutes, or about 45 to 60 minutes. In some embodiments, centrifugation is performed for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, or about 60 minutes. In some embodiments, centrifugation is performed for longer than 60 minutes. In some embodiments, centrifugation of the cell lysate results in a pellet containing inclusion bodies. In some embodiments, centrifugation of the cell lysate results in a pellet free of cell debris. In some embodiments, the cell lysate is a yeast or bacterial cell lysate. In some embodiments, the fusion protein comprises onconase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the onconase is a mutant onconase.

[0262] In some embodiments, the method for purifying inclusion bodies containing a fusion protein includes washing the pellet in a detergent buffer. Detergents are surface-active compounds that reduce the surface tension of water, including nonionic (including, but not limited to, t-octylphenoxypolyethoxyethanol and polyoxyethylenesorbitan), anionic (e.g., sodium dodecyl sulfate), cationic (e.g., cetylpyridinium chloride), and amphoteric agents. Suitable detergents include, but are not limited to, deoxycholic acid, sodium octyl sulfate, sodium tetradecyl sulfate, polyoxyethylene ether, sodium cholate, octylthioglucopyranoside, n-octylglucopyranoside, alkyltrimethylammonium bromide, alkyltrimethylammonium chloride, and bis(2-ethylhexyl) sulfosuccinate. In some embodiments, the detergent buffer includes a nonionic detergent. In some embodiments, the detergent is Triton X-100. In some embodiments, the pellet is washed with detergent at least once, at least twice, or at least three times. In some embodiments, the pellet is washed with detergent more than three times. In some embodiments, the fusion protein comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to two or more oligopeptides by Asp-Pro bonds. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the onconase is a mutant onconase.

[0263] In some embodiments, the purified inclusion bodies are incubated at about 95°C following detergent washes. In some embodiments, the incubation denatures proteins in the inclusion bodies. In some embodiments, the incubation is carried out for at least 1, at least 2, at least 3, at least 5, at least 10, or at least 20 minutes. In some embodiments, the incubation is carried out for 1-20 minutes. In some embodiments, the incubation is between about 90°C and about 100°C.

[0264] In some embodiments, the method for purifying inclusion bodies containing a fusion protein includes washing the pellet in a buffer containing salt. In some embodiments, the buffer has a high salt level. Such a buffer is advantageous for disaggregating and / or refolding the mixture and for maintaining a desired pH value or range. Inorganic salt buffers (e.g., phosphate, carbonate, sodium, among others) and organic salt buffers (e.g., citrate, Tris, MOPS, MES, HEPES, among others) are well known in the art. In some embodiments, the salt buffer comprises NaCl. In some embodiments, the salt buffer comprises at least 0.5 M NaCl. In some embodiments, the salt buffer is at least 0.6 M NaCl, at least 0.7 M NaCl, at least 0.75 M NaCl, or at least 1 M NaCl. In some embodiments, the salt buffer is between 0.1M and 0.6M NaCl, between 0.6M and 0.7M NaCl, between 0.7M and 0.8M NaCl, between 0.8M and 0.9M NaCl, or between 0.9M and 1M NaCl. In some embodiments, the pellet is washed with the buffer at least once, at least twice, or at least three times. In some embodiments, the pellet is washed with the buffer more than three times. In some embodiments, the fusion protein comprises onconase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the onconase is a mutant onconase. In some embodiments, the purified inclusion bodies are incubated at 95°C following washing with the buffer.

[0265] In some embodiments, the method for purifying inclusion bodies containing a fusion protein further comprises washing the pellet in water. Washing the pellet with water may remove or reduce any remaining impurities, as well as any salt or detergent molecules remaining in solution. In some embodiments, the pellet is washed in water at least once, at least twice, or at least three times. In some embodiments, the pellet is washed in water more than three times. In some embodiments, washing the pellet with water produces purified inclusion bodies. In some embodiments, the fusion protein comprises onconase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the onconase is a mutant onconase. In some embodiments, the purified inclusion bodies are incubated at 95°C following the water wash.

[0266] In some embodiments, the method for purifying inclusion bodies containing a fusion protein further comprises solubilizing the inclusion bodies. Solubilization may be performed in the presence or absence of a chaotropic agent (e.g., urea or guanidinium hydrochloride), or in the absence of a chaotropic agent. In some embodiments, the method for purifying inclusion bodies containing a fusion protein further comprises solubilizing the inclusion bodies in a solution without a chaotropic agent. In some embodiments, the method for purifying inclusion bodies containing a fusion protein further comprises solubilizing the inclusion bodies in a solution containing a chemical cleavage reagent. In some embodiments, the method for purifying inclusion bodies containing a fusion protein further comprises solubilizing the inclusion bodies in a solution containing acetic acid or formic acid. In some embodiments, the inclusion bodies are solubilized with acetic acid. In some embodiments, the inclusion bodies comprise a fusion protein comprising onconase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the onconase is a mutant onconase.

[0267] In some embodiments, the method for purifying inclusion bodies containing a fusion protein includes a step of purifying the inclusion bodies. Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC), high performance LC (HPLC), fast flow LC (FPLC), hydroxyapatite chromatography, and lectin chromatography. In some embodiments, the method for purifying inclusion bodies containing a fusion protein further includes a step of purifying the inclusion bodies by chromatography. In some embodiments, the method for purifying inclusion bodies containing a fusion protein does not include a step of purifying the inclusion bodies by chromatography.

[0268] In some embodiments, a method for purifying inclusion bodies containing a fusion protein includes expressing a fusion protein comprising an oligopeptide operably linked to a protein that forms inclusion bodies in cells. The protein that forms inclusion bodies in cells can be any insoluble carrier polypeptide that forms inclusion bodies in cells. In some embodiments, the protein that forms inclusion bodies in cells is onconase. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more polypeptides by a peptide bond. In some embodiments, the peptide bond is susceptible to sequence-specific chemical cleavage. In some embodiments, the oligopeptide is 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond.

[0269] In some embodiments, a method for purifying inclusion bodies containing a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to two or more oligopeptides. In some embodiments, the fusion protein comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more polypeptides, eight or more polypeptides, ten or more polypeptides, fifteen or more polypeptides, or twenty or more oligopeptides operably linked to an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and one or more oligopeptides operably linked to the N-terminus or C-terminus of the onconase protein. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide), wherein one or more oligopeptides are linked to the C-terminus of the onconase. In some embodiments, one or more oligopeptides linked to the C-terminus of onconase have the same amino acid sequence. In some embodiments, one or more oligopeptides linked to the C-terminus of onconase have different amino acid sequences. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide), and one or more oligopeptides are linked to the N-terminus of onconase. In some embodiments, one or more oligopeptides linked to the N-terminus of onconase have the same amino acid sequence. In some embodiments, one or more oligopeptides linked to the N-terminus of onconase have different amino acid sequences. In some embodiments, the onconase is a mutant onconase.

[0270] In some embodiments, a method for purifying inclusion bodies containing fusion proteins includes purifying inclusion bodies containing a fusion polypeptide comprising an oligopeptide operably linked to a protein that forms inclusion bodies in a cell, wherein the operably linked linkage is a chemically cleavable amino acid sequence. In some embodiments, the two or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the inclusion bodies contain a fusion polypeptide comprising one or more oligopeptides that are less than 25 amino acids in length. In some embodiments, the one or more oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the one or more oligopeptides are up to 50 amino acids in length. In some embodiments, the one or more oligopeptides are less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some embodiments, the one or more oligopeptides are between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length. In some embodiments, the fusion polypeptide comprises a cleavable Asp-Pro bond. In some embodiments, the method comprises lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a detergent buffer comprising a non-ionic detergent.

[0271] In some embodiments, a method for purifying inclusion bodies containing a fusion protein includes purifying inclusion bodies containing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) linked to one or more miPEPs. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate one or more miRNA families. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate a plant miRNA family. In some embodiments, the method includes lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a detergent buffer comprising a non-ionic detergent.

[0272] In some embodiments, a method for purifying inclusion bodies containing a fusion protein includes purifying inclusion bodies containing a fusion polypeptide further comprising a linker sequence. In some embodiments, the linker sequence is cleavable by sequence-specific chemical cleavage. In some embodiments, the linker sequence separates one or more oligopeptides and / or the linker sequence separates an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the linker sequence separating one or more oligopeptides has the same or a different sequence from the linker sequence separating the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the inclusion bodies contain a fusion polypeptide comprising a linker sequence comprising the amino acid sequence of SEQ ID NO: 11 separating one or more oligopeptides and / or separating the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a linker sequence comprising an Asp-Pro linker that separates one or more oligopeptides and / or separates an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the method comprises lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a detergent buffer comprising a non-ionic detergent.

[0273] In some embodiments, a method for purifying inclusion bodies containing a fusion protein comprises purifying inclusion bodies containing a fusion polypeptide comprising an onconase polypeptide comprising the amino acid sequence of SEQ ID NO: 1 operably linked by a peptide bond to one or more oligopeptides. In some embodiments, the inclusion bodies contain a fusion polypeptide comprising an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond.

[0274] In some embodiments, a method comprises purifying inclusion bodies comprising a fusion polypeptide comprising a mutant onconase comprising one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1 operably linked to one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide comprising one or more insertions or deletions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising an onconase polypeptide having one or more amino acid substitutions that decrease its susceptibility to chemical cleavage relative to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide having one or more deletions or insertions that decrease its susceptibility to chemical cleavage relative to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide having one or more amino acid substitutions that increase the yield of or promote the formation of inclusion bodies relative to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide having one or more deletions or insertions that increase the yield or facilitate the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method comprises lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a detergent buffer comprising a non-ionic detergent.

[0275] In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:2-10 and 15-22, optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20 A. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:2-10 and 15-22, optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20 A. In some embodiments, the fusion polypeptide comprises an onconase polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs:2-10 and 15-22, and optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in FIG. 20A. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the method comprises lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a detergent buffer comprising a non-ionic detergent.

[0276] In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide with a modified N-terminus and / or C-terminus operably linked to one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO:1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO:1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID NO:1. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the method comprises lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a detergent buffer comprising a non-ionic detergent.

[0277] In some embodiments, a method for purifying inclusion bodies containing a fusion protein comprises purifying inclusion bodies containing a fusion polypeptide comprising a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO: 23 operably linked by a peptide bond to one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond.

[0278] In some embodiments, the method comprises purifying inclusion bodies comprising a fusion polypeptide comprising a mutant TAF12 comprising one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 23 operably linked to one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising one or more insertions or deletions relative to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions that reduce susceptibility to chemical cleavage relative to the TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide having one or more deletions or insertions that reduce susceptibility to chemical cleavage relative to the TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions that increase the yield of, or promote the formation of, inclusion bodies relative to the TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide having one or more deletions or insertions that increase the yield or facilitate the formation of inclusion bodies compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the method comprises lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a detergent buffer comprising a non-ionic detergent.

[0279] In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide having a modified N-terminus and / or C-terminus operably linked to one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus relative to SEQ ID NO:23. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the method comprises lysing cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a detergent buffer comprising a non-ionic detergent.

[0280] IV. Onconase Polypeptides Also provided herein are onconase polypeptides for use as insoluble carrier peptides.

[0281] Onconase, also known as ranpirnase, is an RNAse first identified in the leopard frog (Rana pipiens). Onconase is an approximately 104 amino acid long protein stabilized by four disulfide bridges and undergoes pH-dependent denaturation. Without being limited by theory, acidic conditions promote the denaturation of onconase; the denatured form is highly soluble, and efficient renaturation can only be achieved by methods based on the reversible blocking of cysteine ​​residues. Under neutral conditions (approximately pH 7), onconase tends to aggregate, and high-yield production of onconase can result in the formation of insoluble aggregates such as inclusion bodies, resulting in low to undetectable levels of protein solubility. After aggregation, onconase can be removed from solution by selective precipitation. Furthermore, onconase lacks any aspartic acid-proline and asparagine-glycine sequences, making it resistant to common chemical cleavage strategies.

[0282] In some embodiments, the onconase polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide is a truncated onconase polypeptide. In some embodiments, the truncation is at the N-terminus.

[0283] In some embodiments, the onconase polypeptide has one or more amino acid substitutions that decrease its pI. In some embodiments, the onconase polypeptide has a pI of about 8.0, about 7.5, about 7.0, or about 6.5. In some embodiments, the onconase polypeptide has a neutral pI. In some embodiments, the onconase polypeptide has a pI of less than 7.0.

[0284] In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are conservative amino acid substitutions. In some embodiments, the one or more amino acid substitutions are non-conservative amino acid substitutions. In some embodiments, a fusion protein comprises an onconase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide is inactive.

[0285] In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions, insertions, or deletions compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0286] In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions that increase the yield or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions that increase the yield or promote the formation of inclusion bodies including one or more substitutions of cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, the one or more amino acid substitutions result in a mutant onconase polypeptide that has a reduced number of cationic or polar amino acids compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises one or more deletions or insertions that increase the yield or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more insertions or deletions that increase the yield or promote the formation of inclusion bodies result in a mutant onconase that has a reduced number of cationic or polar amino acids compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0287] In some embodiments, the onconase polypeptide comprises one or more charged or polar amino acid mutations to nonpolar amino acids. In some embodiments, the onconase polypeptide comprises one or more alanine substitutions. In some embodiments, the onconase polypeptide comprises one or more lysine to alanine substitutions. In some embodiments, the onconase polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 alanine substitutions. In some embodiments, the onconase polypeptide comprises no lysine residues. In some embodiments, the onconase polypeptide comprises no arginine residues. In some embodiments, the onconase polypeptide comprises no lysine or arginine residues.

[0288] In some embodiments, the onconase polypeptide comprises a modified N-terminus and / or C-terminus. In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises 11 amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises 11 amino acid substitutions at the N-terminus compared to SEQ ID NO: 1.

[0289] In some embodiments, an onconase polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-10, optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20A. In some embodiments, an onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, an onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, an onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, an onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.In some embodiments, an onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, an onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, an onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, an onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, in each case optionally lacking one, two, three, four, five, or all six C-terminal histidine residues, as shown in Figure 20A.

[0290] In some embodiments, the onconase polypeptide comprises an amino acid sequence tag. In some embodiments, the amino acid sequence tag is a purification amino acid sequence tag. In some embodiments, the amino acid sequence tag is a detection amino acid sequence tag. In some embodiments, the tag is less than 20 amino acids in length, less than 10 amino acids in length, or less than 5 amino acids in length. In some embodiments, the tag is between 5 and 20 amino acids in length. In some embodiments, the tag is a hexahistidine or FLAG tag. In some embodiments, the tag is a biotin tag.

[0291] In some embodiments, the onconase polypeptide has an increased expression level compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. Expression levels can be measured by any standard technique known in the art. For example, the amount of protein (in grams) can be compared to the amount of total cellular protein in grams in a given sample, with the measurement determined as the level of recombinant protein per liter. The level or amount can also be measured in comparison to a known standard, such as a BSA control. Other techniques for measuring protein levels include, but are not limited to, light absorption analysis of purified protein samples, antibody-based detection (e.g., Western blot, FACS, immunofluorescence), activity measurements, and microscopic analysis (e.g., phase contrast, Nomarski interferometry, electron or fluorescence microscopy). The level or activity can also be compared to a known standard (such as a known amount of purified active protein) for more precise quantification.

[0292] Also provided herein are compositions comprising the onconase variant polypeptides described herein.

[0293] V. Fusion Polypeptides Also provided herein are fusion polypeptides comprising a polypeptide capable of forming inclusion bodies and one or more oligopeptides. Any of the inclusion body-forming polypeptides disclosed herein may be used. In some embodiments, the inclusion body-forming polypeptide is an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, the carrier is a mutant carrier. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more oligopeptides. In some embodiments, the one or more oligopeptides are operably linked to the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by a cleavable bond. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond.

[0294] In some embodiments, a fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more polypeptides, eight or more polypeptides, ten or more polypeptides, fifteen or more polypeptides, or twenty or more oligopeptides operably linked to an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide), and one or more oligopeptides are operably linked to the N-terminus and / or C-terminus of the onconase protein. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length.

[0295] In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and one or more oligopeptides operably linked to the N-terminus or C-terminus of the onconase protein. In some embodiments, a fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more polypeptides, eight or more polypeptides, ten or more polypeptides, fifteen or more polypeptides, or twenty or more oligopeptides operably linked to the N-terminus and / or C-terminus of the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and one or more oligopeptides linked to the C-terminus of onconase. In some embodiments, the one or more oligopeptides linked to the C-terminus of onconase have the same amino acid sequence. In some embodiments, the one or more oligopeptides linked to the C-terminus of onconase have different amino acid sequences. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and one or more oligopeptides are linked to the N-terminus of onconase. In some embodiments, the one or more oligopeptides linked to the N-terminus of onconase have the same amino acid sequence. In some embodiments, the one or more oligopeptides linked to the N-terminus of onconase have different amino acid sequences.

[0296] In some embodiments, a fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more oligopeptides by a cleavable peptide bond. The cleavable bond can be cleaved by an enzymatic or chemical agent. Cleavage of the peptide bond can separate the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the oligopeptide and / or the oligopeptides from each other. In some embodiments, the peptide bond can be cleaved by enzymatic cleavage. In some embodiments, the peptide bond can be cleaved by sequence-specific chemical cleavage. In some embodiments, (i) the peptide bond comprises methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises cysteine ​​and the sequence-specific chemical cleavage uses NTCB. In some embodiments, one or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by a peptide bond comprising an Asp-Pro bond and the sequence-specific chemical cleavage uses formic acid or acetic acid.

[0297] In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more oligopeptides that are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the one or more oligopeptides are less than 25 amino acids in length. In some embodiments, the one or more oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the one or more oligopeptides are up to 50 amino acids in length. In some embodiments, the one or more oligopeptides are less than 50, 45, 40, 35, 30, 25, or 20 amino acids in length. In some embodiments, the one or more oligopeptides are between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length. In some embodiments, the one or more oligopeptides are operably linked to each other by an Asp-Pro bond. In some embodiments, the operably linkage between the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the one or more oligopeptides comprises an Asp-Pro bond. In some embodiments, the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is a mutant onconase.

[0298] In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more oligopeptides comprising the amino acid sequence of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the onconase is a mutant onconase. In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0299] In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) linked to one or more micropeptides (miPEPs). In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a miRNA. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a plant miRNA, a fungal miRNA, or a metazoan miRNA. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a plant miRNA. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a plant miRNA selected from the group consisting of the miR156, miR159 / 319, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR390, miR393, miR394, miR295, miR396, miR397, miR398, miR408, miR403, miR437, miR444, and miR445 families. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that has microbe inhibitory activity. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the onconase is a mutant onconase. In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0300] In some embodiments, the fusion polypeptide further comprises a linker sequence between one or more oligopeptides and / or between the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the oligopeptide. The linker sequence may function as a spacer peptide separating the one or more oligopeptides and / or the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the oligopeptide. In some embodiments, the linker sequence provides a cleavable peptide bond. For example, the linker sequence may provide a cleavable peptide bond by incorporation of an amino acid sequence that directs sequence-specific cleavage of the fusion polypeptide. In some embodiments, the linker sequence is cleavable by sequence-specific chemical cleavage. In some embodiments, the fusion polypeptide comprises a linker sequence separating one or more oligopeptides and / or a linker sequence separating the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, the linker sequence separating the one or more oligopeptides has the same or a different sequence as the linker sequence separating the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, the fusion polypeptide comprises a linker sequence comprising the amino acid sequence of SEQ ID NO: 11 that separates the one or more oligopeptides and / or separates the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, the fusion polypeptide comprises a linker sequence comprising an Asp-Pro linker that separates the one or more oligopeptides and / or separates the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, sequence-specific chemical cleavage of the linker sequence releases the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the one or more oligopeptides and / or the one or more oligopeptides from each other.

[0301] In some embodiments, the fusion polypeptide further comprises one or more amino acid sequence tags. In some embodiments, the amino acid sequence tag does not affect the solubility of the fusion polypeptide. In some embodiments, the amino acid sequence tag has a neutral pI. In some embodiments, the one or more amino acid sequence tags are purification amino acid sequence tags. Such tag peptides may include, but are not limited to, glutathione-S-transferase (GST), polyhistidine, maltose-binding protein (MBP), avidin, biotin, streptavidin, histidine (His) tag (e.g., His-6X tag), V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, thioredoxin (Trx) tag, and ligands for cellular receptors (e.g., insulin receptor ligands). In some embodiments, the one or more amino acid sequence tags are detection or reporter amino acid sequence tags. Examples of reporter amino acid sequence tags include, but are not limited to, horseradish peroxidase (HRP), chromamphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, and fluorescent proteins (e.g., GFP, CFP, YFP, BFP, etc.). In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus and / or C-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) or one or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the N-terminus of one or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the N-terminus of one or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of one oligopeptide and the N-terminus of another oligopeptide.

[0302] In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising the amino acid sequence of SEQ ID NO: 1 operably linked by a peptide bond to one or more oligopeptides. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide is a mutant onconase polypeptide.

[0303] In some embodiments, a fusion polypeptide comprises a mutant onconase polypeptide operably linked to one or more oligopeptides by peptide bonds. In some embodiments, the mutant onconase comprises one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO: 1. The one or more insertions or deletions can be of any amino acid length.

[0304] In some embodiments, a fusion protein comprises an onconase polypeptide having one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide having one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0305] In some embodiments, a fusion protein comprises an onconase polypeptide having one or more amino acid substitutions that increase the yield of or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions that increase the yield of or promote the formation of inclusion bodies comprise substitutions of one or more cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, the one or more amino acid substitutions result in a mutant onconase having a reduced number of cationic or polar amino acids compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an onconase polypeptide having one or more deletions or insertions that increase the yield of or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more insertions or deletions that increase the yield of or promote the formation of inclusion bodies result in a mutant onconase having a reduced number of cationic or polar amino acids compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0306] In some embodiments, a fusion protein comprises an onconase polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-10 or 15-22 operably linked to one or more oligopeptides, optionally with one, two, three, four, five, or all six C-terminal histidine residues deleted, as shown in Figure 20 A. In some embodiments, a fusion protein comprises an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-10 or 15-22, optionally with one, two, three, four, five, or all six C-terminal histidine residues deleted, as shown in Figure 20A. In some embodiments, the onconase polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NOs: 2-10 or 15-22, and optionally has one, two, three, four, five, or all six C-terminal histidine residues deleted as shown in FIG. 20A. In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises one or more deletions or insertions compared to SEQ ID NO: 1. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0307] In some embodiments, a fusion polypeptide comprises an onconase polypeptide having a modified N-terminus and / or C-terminus operably linked to one or more oligopeptides. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO: 1. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO: 1. In some embodiments, a fusion polypeptide comprises an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus relative to SEQ ID NO: 1. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the operably linkage comprises an Asp-Pro bond.

[0308] In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO: 23 operably linked to one or more oligopeptides by peptide bonds. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NO: 23. In some embodiments, the TAF12 polypeptide is a mutant TAF12 polypeptide.

[0309] In some embodiments, a fusion polypeptide comprises a mutant TAF12 polypeptide operably linked to one or more oligopeptides by peptide bonds. In some embodiments, the mutant TAF12 comprises one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO: 23. The one or more insertions or deletions may be of any amino acid length.

[0310] In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23.

[0311] In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions that increase the yield of or promote the formation of inclusion bodies compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more amino acid substitutions that increase the yield of or promote the formation of inclusion bodies comprise substitutions of one or more cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, the one or more amino acid substitutions result in a mutant TAF12 polypeptide having a reduced number of cationic or polar amino acids compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, a fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions that increase the yield of or promote the formation of inclusion bodies compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more insertions or deletions that increase the yield of or promote the formation of inclusion bodies result in a mutant TAF12 polypeptide having a reduced number of cationic or polar amino acids compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0312] In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions compared to SEQ ID NO: 23. In some embodiments, the TAF12 polypeptide comprises one or more deletions or insertions compared to SEQ ID NO: 23. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0313] In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide having a modified N-terminus and / or C-terminus operably linked to one or more oligopeptides. In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, a fusion polypeptide comprises a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus relative to SEQ ID NO:23. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operably linkage comprises an Asp-Pro bond.

[0314] In some embodiments, the fusion polypeptide is expressed in a cell or cell culture. Any cell or cell culture known to produce inclusion bodies is suitable for expressing the fusion polypeptide. In some embodiments, the fusion polypeptide is expressed in bacteria. In some embodiments, the bacteria is a strain of Escherichia coli or Vibrio natrigens. In some embodiments, the bacteria is an E. coli strain. In some embodiments, the bacterial cell is a strain lacking Lon and ompT protease function (e.g., a BL21 E. coli strain). In some embodiments, the fusion polypeptide is a yeast cell. In some embodiments, the yeast cell is a Pichia pastoris strain. In some embodiments, the fusion polypeptide is produced by any of the methods disclosed herein.

[0315] VI. Oligopeptides Provided herein are oligopeptides comprising an active amino acid sequence. In some embodiments, the active amino acid sequence is 4-50 or 5-30 amino acids in length. In some embodiments, the active amino acid sequence is a micropeptide (miPEP). In some embodiments, the oligopeptide comprises the active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid. miPEPs are short peptides (7-44 amino acids in length) defined by one or several short open reading frames located in the pri-microRNA sequence of a specific miRNA member. Without being limited by theory, it is believed that miPEPs target their encoding microRNAs at the transcriptional level and regulate the expression of the target pri-microRNA, thereby up-regulating or down-regulating transcription. miPEPs may target miRNAs involved in various processes, such as miRNAs involved in immunity and susceptibility to pathogens (e.g., microorganisms), organogenesis, stress response, embryonic development, etc.

[0316] In some embodiments, the oligopeptide comprises a miPEP sequence that regulates a miRNA. Each miPEP may regulate one or more members of a specific miRNA family and in a specific species. The miPEP sequence may regulate plant miRNAs and metazoans. In some embodiments, the miPEP sequence regulates one or more members of a specific miRNA family. In some embodiments, the miPEP sequence regulates a plant miRNA. Representative plant microRNA families that can be regulated by miPEP include, but are not limited to, the plant miRNA families miR156, miR159 / 319, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR390, miR393, miR394, miR295, miR396, miR397, miR398, miR408, miR403, miR437, miR444, and miR445. In some embodiments, the miPEP sequence comprises an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the miPEP sequence has microbe-inhibiting activity.

[0317] In some embodiments, the oligopeptide comprises an active amino acid sequence having microbial inhibitory activity. In other embodiments, the oligopeptide comprises a peptide microbial inhibitor. In some embodiments, the oligopeptide inhibits a microbial organism. In some embodiments, the microbial organism is a virus, bacterium, fungus, amoeba, or eukaryote. Exemplary microbial targets that may be inhibited by the oligopeptides include Venturia, Podosphaera, Erysiphe, Monolinia, Mycosphaerella, Uncinula, Hemileia, Rhizoctonia, Puccinia, Botrytis, Helminthos, and the like. Helminthosporium, Rhynchosporium, Fusarium, Septoria, Cercospora, Alternaria, Pyricularia, Pseudocercosporella, Phytophthora, Peronospora Bremia, Pythium, Plasmopara, Scleropthora, Peronosclerospora, Physopella, Cercospora, Colletotrichum, Gibberell, Exserohilum, Bacillus, Trichoderma, Kabatiellu, Bipolaris, Pseudomonas, Pseudomonas Erwinia, Mycoplasma, and Rickettsia.In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0318] In some embodiments, the oligopeptide is less than 25 amino acids in length. In some embodiments, the oligopeptide is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the oligopeptide is up to 50 amino acids in length. In some embodiments, the oligopeptide is less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some embodiments, the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length. In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0319] In some embodiments, the oligopeptide comprises an active amino acid sequence that is 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the active amino acid sequence is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the oligopeptide is up to 50 amino acids in length. In some embodiments, the active amino acid sequence is less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some embodiments, the active amino acid sequence is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length. In some embodiments, the oligopeptide comprises a tag comprising the active amino acid sequence and additional amino acids. In some embodiments, the tag is a remnant of cleavage of the linker. In some embodiments, the tag is located N- or C-terminal to the active amino acid sequence. In some embodiments, the oligopeptide comprises tags at both the N- and C-termini.

[0320] In some embodiments, the oligopeptide comprises the amino acid sequence of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, one or more oligopeptides comprise an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acids in length. In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0321] In some embodiments, the oligopeptides provided herein are produced by cleavage of a concatemer of multiple oligopeptides that are linked together and expressed as a single polypeptide chain. In some embodiments, the cleavage is of a homoconcatemer. In some embodiments, the cleavage is of a heteroconcatemer. In some embodiments, the cleavage results in a short (one to two) amino acid tag at the N- and / or C-terminus of the oligopeptide.

[0322] In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal and / or C-terminal residue. The N-terminal and / or C-terminal residue may correspond to a cleavage tag. For example, the residue may be part of a peptide bond that is cleaved by a chemical reaction (acid cleavage) or an enzymatic reaction. In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal proline, methionine, tryptophan, glycine, or cysteine, and / or a C-terminal methionine, aspartic acid, cysteine, asparagine, or tryptophan. In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acids in length. In some embodiments, the active amino acid sequence is a miPEP.

[0323] VII. Composition Provided herein are compositions comprising one or more oligopeptides produced by any of the disclosed methods. In some embodiments, the one or more oligopeptides comprise an active amino acid sequence. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the one or more oligopeptides comprise an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0324] In some embodiments, the compositions provided herein are produced by cleavage of a concatemer polypeptide. In some embodiments, the concatemer polypeptide is a heteroconcatemer. In some embodiments, the concatemer polypeptide is a homoconcatemer. In some embodiments, the compositions comprise a single type of active oligopeptide produced by cleavage of a homoconcatemer. In some embodiments, the compositions comprise multiple active oligopeptides produced by cleavage of a heteroconcatemer. Various ratios of peptides in the compositions are contemplated, such as 1:1, 1:2, 1:3, 1:4, 1:1:1, etc.

[0325] In some embodiments, the composition is formulated as a liquid, gel, emulsion, suspension, encapsulation, solid, powder, aerosol, paste, coating, spray, soil drench, microcapsule, emulsifiable concentrate, or granule. In some embodiments, the composition is formulated for agricultural use. In some embodiments, the composition is formulated as a seed treatment, foliar spray, foliar drench, ready-to-use (RTU) formulation, produce coating, suspension concentrate, tank mix, aerosol, root dip, soil treatment, dip formulation, irrigation formulation, or sprinkler formulation. In some embodiments, the composition is formulated to be applied to plants. In some embodiments, the composition is formulated to be applied to one or more of seeds, roots, tubers, fruits, leaves, bulbs, rhizomes, or flowers. In some embodiments, the composition can be applied to plants by foliar spray, foliar drench, drip irrigation, coating, mixing, injecting, dusting, misting, soil drenching, fumigation, soil injection, percolation irrigation, sprinklers, or manual irrigation. In some embodiments, the composition is formulated to be applied to plants by foliar spray. In some embodiments, compositions formulated for agricultural use comprise one or more oligopeptides. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the one or more oligopeptides comprise an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0326] In some embodiments, the composition is formulated as a pharmaceutically acceptable composition. In some embodiments, the pharmaceutically acceptable composition is formulated as a liquid, emulsion, liquefied drops, spray, foam, granules, fine granules, powder, capsule, pill, paste, tablet, chewable, injection, suppository, cream, shampoo, rinse, resin, smoke, or bait. In some embodiments, the pharmaceutically acceptable composition is formulated for administration to a non-human animal. In some embodiments, the pharmaceutically acceptable composition is formulated for administration to a human. In some embodiments, the pharmaceutically acceptable composition comprises one or more oligopeptides. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the one or more oligopeptides comprise an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0327] In some embodiments, the composition comprises one or more oligopeptides and a carrier. In some embodiments, the carrier is an agriculturally acceptable carrier. In some embodiments, the agriculturally acceptable carrier comprises a solid carrier, a liquid carrier, a gel carrier, a suspension, or an emulsion. In some embodiments, the agriculturally acceptable carrier comprises an adjuvant, an inactive ingredient, a dispersant, a surfactant, an emulsifier, a thickener, a wetting agent, a fertilizer, a mineral, a solvent, a tackifier, a binder, or a stabilizer. In some embodiments, the composition comprises one or more oligopeptides and a pharmaceutically acceptable carrier. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the one or more oligopeptides comprise an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0328] In some embodiments, the composition comprises one or more oligopeptides comprising an active amino acid sequence. In some embodiments, the active amino acid sequence is a micropeptide (miPEP). In some embodiments, the oligopeptide comprises a miPEP sequence that regulates a miRNA. In some embodiments, the miPEP sequence regulates one or more members of a specific miRNA family. In some embodiments, the miPEP sequence regulates a plant miRNA. Representative plant microRNA families that may be regulated by miPEPs include, but are not limited to, the plant miRNA families miR156, miR159 / 319, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR390, miR393, miR394, miR295, miR396, miR397, miR398, miR408, miR403, miR437, miR444, and miR445. In some embodiments, a composition comprises a miPEP with microbial inhibitory activity. In some embodiments, a composition comprises a miPEP that inhibits viruses, bacteria, fungi, amoebas, or eukaryotes. In some embodiments, a miPEP comprises an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length.

[0329] In some embodiments, the composition comprises one or more oligopeptides comprising the amino acid sequence of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the composition comprises two or more oligopeptides. In some embodiments, the composition comprises three or more oligopeptides.

[0330] In some embodiments, the compositions comprise one or more oligopeptides comprising an active amino acid sequence and an N-terminal and / or C-terminal residue. The N-terminal and / or C-terminal residue may correspond to a cleavage tag. In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal proline, methionine, tryptophan, glycine, or cysteine, and / or a C-terminal methionine, aspartic acid, cysteine, asparagine, or tryptophan. In some embodiments, the compositions comprise one or more oligopeptides comprising an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0331] VIII. Nucleic acids Nucleic acids encoding any of the fusion polypeptides, polypeptides, insoluble carrier polypeptides (e.g., onconase polypeptides or TAF12 polypeptides), or oligopeptides disclosed herein are also provided. In some embodiments, the nucleic acid comprises sequence elements that enhance expression of the encoded polypeptide in a cell. For example, the nucleic acid may comprise an origin of replication that, when incorporated into a vector, directs a copy number high enough to result in expression of the encoded polypeptide. In some embodiments, the nucleic acid comprises a promoter that regulates expression of the fusion polypeptide. In some embodiments, the promoter is a bacterial promoter or a yeast promoter. In some embodiments, the promoter is a constitutive promoter or an inducible promoter. In some embodiments, the promoter is a strong promoter. In some embodiments, the promoter drives expression of the encoded polypeptide in inclusion bodies.

[0332] Nucleic acids of the present disclosure may be prepared using any technique known in the art, including, but not limited to, cloning, DNA isolation, amplification and purification, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques such as gel electrophoresis and chromatography. Some standard techniques are described in Ausubel et al. (1992) Current Protocols in Molecular Biology, Green / Wiley, New York, NY; Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, NY; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY; Wu (eds.) (1993) Meth. Enzymol. 218, Part I; Wu (eds.) (1979) Meth. Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (eds.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol.I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York; and Ausubel et al. (1992) Current Protocols in Molecular Biology, Greene / Wiley, New York, NY. Abbreviations and nomenclature, where used, are considered standard in the art and commonly used in professional journals such as those cited herein.

[0333] Also provided herein are vectors containing a nucleic acid encoding any of the fusion polypeptides, insoluble carrier polypeptides (eg, onconase polypeptides or TAF12 polypeptides), or oligopeptides disclosed herein.

[0334] A "vector" is a nucleic acid capable of carrying another nucleic acid. A vector may be, for example, a plasmid, virus, cosmid, or phage. An "expression vector" is a vector that, when present in the appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector. An example of a vector is one that is capable of autonomous replication and expression of structural gene products present in DNA segments to which it is operably linked. Thus, vectors can include replicons and selectable markers as previously described. Vectors include, but are not necessarily limited to, expression vectors. In some embodiments, the vector is a bacterial vector. In some embodiments, the vector is a yeast vector.

[0335] Examples of expression vectors that can be used in prokaryotic host cells include the cloning vector pET plasmid (Novagen, Madison, Wis., USA) or expression vectors derived from commercially available plasmids such as pBR322 (ATCC 37017). The pBR322 vector contains genes for ampicillin and tetracycline resistance, thus providing a simple means for identifying transformed cells. To construct an expression vector using pBR322, a suitable promoter and a DNA sequence encoding one or more polypeptides of the present invention are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM-1 (Promega Biotec, Madison, Wis., USA). Other commercially available vectors include vectors specifically designed for the expression of proteins, and these vectors may include the pMAL-p2 and pMAL-c2 vectors (New England Biolabs, Beverly, Mass., USA), which are used for the expression of proteins fused to maltose-binding protein.

[0336] In yeast, a number of vectors containing constitutive or inducible promoters may be used. Review articles include Current Protocols in Molecular Biology, Vol. 2, ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Bitter et al., Expression and Secretion Vectors for Yeast, in Methods in Enzymology, eds. Wu & Grossman, 1987, Acad. Press, NY, Vol. 153, pp. 516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press, Wash., DC, Ch. 3 (1986); Bitter, Heterologous Gene Expression in Yeast, Methods in Enzymology, eds. Berger & Kimmel, Acad. Press, NY, Vol. 152, pp. 673-684 (1987); and The Molecular Biology of the Yeast Saccharomyces, eds. Strathem et al., Cold Spring Harbor Press, Vols. I and II (1982). Constitutive yeast promoters such as ADH1 or LEU2 or inducible promoters such as GAL4 may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, ed. D. M. Glover, IRL Press, Wash., DC (1986)). Alternatively, vectors that facilitate integration of foreign DNA sequences into the yeast or bacterial chromosome may be used.

[0337] Recombinant prokaryotic host cell expression vectors include the bacteriophage T7 promoter (Studier and Moffatt, J. Mol. Biol. 189: 113 (1986)), β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature 281:544 (1979)), tryptophan (tap) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); EP-A-36776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; p. 412 (1982)). A particularly useful prokaryotic host cell expression system uses the phage λ PL promoter and cl857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection (ATCC) that incorporate derivatives of the PL promoter include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).

[0338] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a protein that forms inclusion bodies in cells operably linked to one or more oligopeptides by peptide bonds. In some embodiments, the inclusion body tag is an onconase polypeptide. In some embodiments, the protein that forms inclusion bodies in cells is onconase. In some embodiments, the onconase is a mutant onconase.

[0339] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more polypeptides by peptide bonds. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a fusion polypeptide comprising two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more polypeptides, eight or more polypeptides, ten or more polypeptides, fifteen or more polypeptides, or twenty or more oligopeptides operably linked to an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, the one or more oligopeptides are operably linked to the N-terminus and / or C-terminus of onconase. In some embodiments, the one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion polypeptide encoded by the nucleic acid comprises a cleavable Asp-Pro bond.

[0340] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more polypeptides by a peptide bond capable of sequence-specific chemical cleavage. In some embodiments, (i) the peptide bond comprises a methionine, and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises a tryptophan, and the sequence-specific chemical cleavage uses BNPS skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond, and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond, and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises a cysteine, and the sequence-specific chemical cleavage uses NTCB. In some embodiments, the one or more oligopeptides are operably linked to the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) by a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage uses formic acid or acetic acid.

[0341] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising one or more oligopeptides that are less than 25 amino acids in length. In some embodiments, the one or more oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the one or more oligopeptides are up to 50 amino acids in length. In some embodiments, the one or more oligopeptides are less than 50, 45, 40, 35, 30, 25, or 20 amino acids in length. In some embodiments, the one or more oligopeptides are between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

[0342] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising onconase operably linked to one or more oligopeptides having the amino acid sequence of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise at least one of the amino acid sequences of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the onconase is a mutant onconase. In some embodiments, the one or more oligopeptides, when released from the fusion polypeptide, comprise an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the nucleic acid further comprises a linker sequence between the one or more oligopeptides and / or between the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the oligopeptide.In some embodiments, the linker sequence is cleavable by sequence-specific chemical cleavage.

[0343] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more miPEPs. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a miRNA. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates one or more miRNA families. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a plant miRNA. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that has microbe-inhibiting activity.

[0344] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide comprising the amino acid sequence of SEQ ID NO: 1 operably linked to one or more oligopeptides by peptide bonds. In some embodiments, the onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO: 1.

[0345] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide with one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide with one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0346] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide having one or more amino acid substitutions, or one or more deletions or insertions, that increase the yield of or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide having the amino acid sequence of any one of SEQ ID NOs: 2-10, optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20A. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs:2-10 and 15-22, optionally with one, two, three, four, five, or all six C-terminal histidine residues deleted, as shown in Figure 20 A. In some embodiments, the onconase polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs:2-10 and 15-22, optionally with one, two, three, four, five, or all six C-terminal histidine residues deleted, as shown in Figure 20A.

[0347] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide with a modified N-terminus and / or C-terminus operably linked to one or more oligopeptides. In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID NO: 1. In some embodiments, the nucleic acid further comprises a linker sequence between one or more oligopeptides and / or between the onconase polypeptide and the oligopeptide. In some embodiments, the linker sequence is cleavable by sequence-specific chemical cleavage.

[0348] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO:23 operably linked to one or more oligopeptides by peptide bonds. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs:23. In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO:23. In some embodiments, the TAF12 polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO:23.

[0349] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide with one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide with one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23.

[0350] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions, or one or more deletions or insertions, that increase the yield of or promote the formation of inclusion bodies compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23.

[0351] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide with a modified N-terminus and / or C-terminus operably linked to one or more oligopeptides. In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus relative to SEQ ID NO:23. In some embodiments, the nucleic acid further comprises a linker sequence between one or more oligopeptides and / or between the TAF12 polypeptide and the oligopeptide. In some embodiments, the linker sequence is cleavable by sequence-specific chemical cleavage.

[0352] In some embodiments, the nucleic acid encodes an oligopeptide. In some embodiments, the nucleic acid encodes an oligopeptide comprising an active amino acid sequence. In some embodiments, the active amino acid sequence is a micropeptide (miPEP). In some embodiments, the nucleic acid encodes an oligopeptide comprising a miPEP sequence that regulates a miRNA. In some embodiments, the miPEP sequence regulates one or more members of a specific miRNA family. In some embodiments, the miPEP sequence regulates a plant miRNA. In some embodiments, the miPEP sequence regulates a plant miRNA family selected from the group consisting of miR156, miR159 / 319, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR390, miR393, miR394, miR295, miR396, miR397, miR398, miR408, miR403, miR437, miR444, and miR445. In some embodiments, the miPEP comprises an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0353] In some embodiments, the nucleic acid encodes a miPEP that has microbe-inhibiting activity.

[0354] In some embodiments, the nucleic acid encodes an oligopeptide comprising the amino acid sequence of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising at least one of the amino acid sequences of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the oligopeptide is 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the oligopeptide comprises an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0355] In some embodiments, the nucleic acid encodes an onconase polypeptide. In some embodiments, the onconase is a mutant onconase. In some embodiments, the nucleic acid encodes an onconase polypeptide comprising the amino acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid encodes an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the nucleic acid encodes an onconase polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO:1. In some embodiments, the nucleic acid encodes an onconase polypeptide comprising one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid encodes an onconase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO:1. In some embodiments, the one or more amino acid substitutions, or one or more deletions or insertions, decrease the susceptibility of the onconase polypeptide to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions, or one or more insertions or deletions, result in a mutant onconase that has an increased yield of, or an increased ability to form, inclusion bodies in a cell compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, a nucleic acid encodes an onconase polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-10 and 15-22, optionally with one, two, three, four, five, or all six C-terminal histidine residues deleted, as shown in Figure 20A.In some embodiments, the nucleic acid encodes an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 2-10 and 15-22, and optionally one, two, three, four, five, or all six C-terminal histidine residues deleted, as shown in Figure 20A. In some embodiments, the nucleic acid encodes an onconase polypeptide having at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401.

[0356] In some embodiments, the nucleic acid encodes an onconase polypeptide comprising a modified N-terminus and / or C-terminus. In some embodiments, the nucleic acid encodes an onconase polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO: 1. In some embodiments, the nucleic acid encodes an onconase polypeptide comprising 1 ... relative to SEQ ID NO: 1.

[0357] In some embodiments, the nucleic acid encodes a TAF12 polypeptide. In some embodiments, the TAF12 is a mutant TAF12. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO:23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO:23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO:23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO:23. In some embodiments, the one or more amino acid substitutions, or one or more deletions or insertions, reduce the susceptibility of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more amino acid substitutions, or one or more insertions or deletions, result in a mutant TAF12 that has an increased yield of, or an increased ability to form, inclusion bodies in a cell compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, a nucleic acid encodes a TAF12 polypeptide having at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401.

[0358] In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising a modified N-terminus and / or C-terminus. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO: 23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising 1 ... relative to SEQ ID NO: 23.

[0359] In some embodiments, the nucleic acid encodes an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) comprising an amino acid sequence tag. In some embodiments, the amino acid sequence tag is a purification amino acid sequence tag. In some embodiments, the amino acid sequence tag is a detection amino acid sequence tag. In some embodiments, the onconase is a mutant onconase.

[0360] IX.Cells Also provided are cells that express any of the fusion polypeptides, oligopeptides, tagged miPEPs, or insoluble carrier polypeptides (e.g., onconase polypeptides or TAF12 polypeptides) described herein. In some embodiments, the cells are bacterial cells or yeast cells. In some embodiments, the cells are host cells.

[0361] In some embodiments, the cell is a bacterial cell, a yeast cell, a plant cell, or a metazoan cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the bacterium is an E. coli strain. In some embodiments, the cell is a cell capable of forming inclusion bodies. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is an algae cell.

[0362] Microbial host cells useful in the present invention include, for example, Enterobacteriaceae (e.g., Escherichia and Salmonella) and bacteria such as Bacillus, Acinetobacter, Streptomyces, Methylobacter, Rhodococcus, and Pseudomonas; Cyanobacteria such as Rhodobacter and Synechocystis; a); yeasts such as Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Yarrowia, and Torulopsis; and filamentous fungi such as Aspergillus and Arihroboirys, and algae, but are not limited to these.

[0363] In some embodiments, the cells comprise one or more modifications to increase expression of the fusion protein. In some embodiments, the cells comprise one or more protease mutations or deletions. In some embodiments, the cells comprise a mutation or deletion in the OmpT or Lon gene.

[0364] In some embodiments, the host cell comprises at least one copy of a nucleic acid sequence encoding the fusion polypeptide. At least one copy of the nucleic acid sequence encoding the fusion polypeptide enzyme can be present in the chromosome of a prokaryotic (bacterial) cell or in one chromosome of a eukaryotic cell. Alternatively, at least one copy of the nucleic acid sequence encoding the fusion polypeptide can be present in a vector or plasmid present in the cell. The host cell can be a prokaryotic or eukaryotic cell, as described above. If it is a prokaryotic cell, it can be a bacterial cell. If it is a eukaryotic cell, it can be a yeast cell, a plant cell, or an animal cell. Suitable host cells are described herein.

[0365] In some embodiments, the cells express a fusion polypeptide comprising an insoluble carrier polypeptide, which forms inclusion bodies, and the insoluble carrier polypeptide is operably linked to one or more oligopeptides. In some embodiments, the insoluble carrier polypeptide, which forms inclusion bodies, is an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is a mutant insoluble carrier polypeptide (e.g., a mutant onconase polypeptide or a mutant TAF12 polypeptide). In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the one or more oligopeptides are operably linked to the N-terminus and / or C-terminus of the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide). In some embodiments, the operably linkage comprises an Asp-Pro bond.

[0366] In some embodiments, the cells express a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more oligopeptides comprising the amino acid sequence of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise at least one of the amino acid sequences of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the one or more oligopeptides, when released from the fusion polypeptide, comprise an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.In some embodiments, the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is a mutant insoluble carrier polypeptide (e.g., a mutant onconase polypeptide or a mutant TAF12 polypeptide). In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0367] In some embodiments, the cells express a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) operably linked to one or more micropeptides (miPEPs). In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a miRNA. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates one or more members of a miRNA family. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a plant miRNA. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that regulates a plant miRNA selected from the group consisting of the miR156, miR159 / 319, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR390, miR393, miR394, miR295, miR396, miR397, miR398, miR408, miR403, miR437, miR444, and miR445 families. In some embodiments, the one or more oligopeptides comprise a miPEP sequence that has microbial inhibitory activity. In some embodiments, the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) is a mutant insoluble carrier polypeptide (e.g., a mutant onconase polypeptide or a mutant TAF12 polypeptide). In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0368] In some embodiments, the cells express fusion polypeptides further comprising a linker sequence between one or more oligopeptides and / or between the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) and the oligopeptide. In some embodiments, a linker sequence separates the one or more oligopeptides and / or a linker sequence separates the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, the linker sequence separating the one or more oligopeptides has the same or a different sequence as the linker sequence separating the insoluble carrier polypeptide (e.g., an onconase polypeptide or a TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, the cells express fusion polypeptides further comprising one or more amino acid sequence tags.

[0369] In some embodiments, the cells express a fusion polypeptide comprising an insoluble carrier polypeptide comprising an onconase polypeptide. In some embodiments, the onconase polypeptide comprises the amino acid sequence of SEQ ID NO: 1 operably linked to one or more oligopeptides by peptide bonds. In some embodiments, the onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions, or one or more insertions or deletions, compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions, or the one or more insertions or deletions, decrease the susceptibility of the onconase to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions, or the one or more insertions or deletions, result in an onconase polypeptide that has an increased yield or a greater ability to form inclusion bodies in a cell compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0370] In some embodiments, the onconase polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:2-10 and 15-22 operably linked to one or more oligopeptides, and optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20 A. In some embodiments, the cells express a fusion polypeptide comprising an onconase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs:2-10 and 15-22, and optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20A. In some embodiments, the onconase polypeptide comprises at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 1-10 and 15-22, and optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20A. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0371] In some embodiments, the onconase polypeptide has a modified N-terminus and / or C-terminus operably linked to one or more oligopeptides. In some embodiments, the cell expresses a fusion polypeptide comprising an onconase polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, the cell expresses a fusion polypeptide comprising an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, the cell expresses a fusion polypeptide comprising an onconase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID NO: 1. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the operably linkage comprises an Asp-Pro bond.

[0372] In some embodiments, the cells express a fusion polypeptide comprising an insoluble carrier polypeptide comprising a TAF12 polypeptide. In some embodiments, the TAF12 polypeptide comprises the amino acid sequence of SEQ ID NO:23 operably linked to one or more oligopeptides by peptide bonds. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO:23. In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions, or one or more insertions or deletions, compared to the amino acid sequence of SEQ ID NO:23. In some embodiments, the one or more amino acid substitutions, or the one or more insertions or deletions, decrease the susceptibility of TAF12 to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more amino acid substitutions, or the one or more insertions or deletions, result in a TAF12 polypeptide that has increased yield or a greater ability to form inclusion bodies in a cell compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

[0373] In some embodiments, the TAF12 polypeptide has a modified N-terminus and / or C-terminus operably linked to one or more oligopeptides. In some embodiments, the cell expresses a fusion polypeptide comprising a TAF12 polypeptide comprising one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, the cell expresses a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO:23. In some embodiments, the cell expresses a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus relative to SEQ ID NO:23. In some embodiments, the one or more oligopeptides are 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the operably linkage comprises an Asp-Pro bond.

[0374] In some embodiments, the cells express an oligopeptide. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acids in length. In some embodiments, the cells express an oligopeptide comprising an active amino acid sequence. In some embodiments, the active amino acid sequence is a miPEP sequence. In some embodiments, the miPEP sequence regulates a miRNA. In some embodiments, the miPEP sequence regulates a plant miRNA. In some embodiments, the miPEP comprises an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0375] In some embodiments, the cell expresses an oligopeptide comprising the amino acid sequence of any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the oligopeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the oligopeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the cell expresses an oligopeptide comprising at least one of the amino acid sequences of any one of SEQ ID NOs: 12-14 and 31-2401.

[0376] In some embodiments, the oligopeptide is 4 to 50 or 5 to 30 amino acids in length. In some embodiments, the oligopeptide comprises an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid.

[0377] In some embodiments, the cells express an onconase polypeptide. In some embodiments, the onconase polypeptide is a mutant onconase polypeptide. In some embodiments, the onconase polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO: 1. In some embodiments, the cells express an onconase polypeptide comprising one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cells express an onconase polypeptide that comprises one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cells express an onconase polypeptide that comprises one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0378] In some embodiments, the cells express an onconase polypeptide that includes one or more amino acid substitutions, or one or more insertions or deletions, that increase the yield of or promote the formation of inclusion bodies compared to an onconase polypeptide having the amino acid sequence of SEQ ID NO:11.

[0379] In some embodiments, the cells express an onconase polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:2-10 and 15-22, optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20 A. In some embodiments, the onconase polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs:2-10 and 15-22, optionally lacking one, two, three, four, five, or all six C-terminal histidine residues as shown in Figure 20 A. In some embodiments, the onconase polypeptide comprises at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the onconase polypeptide comprises a modified N-terminus and / or C-terminus. In some embodiments, the onconase polypeptide comprises one or more amino acid substitutions at the N-terminus and / or C-terminus compared to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises an 11 amino acid substitution at the N-terminus compared to SEQ ID NO: 1. In some embodiments, the onconase polypeptide comprises a sequence tag.

[0380] In some embodiments, the cells express a TAF12 polypeptide. In some embodiments, the TAF12 polypeptide is a mutant TAF12 polypeptide. In some embodiments, the TAF12 polypeptide comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to SEQ ID NO:23. In some embodiments, the cells express a TAF12 polypeptide comprising one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO:23. In some embodiments, the TAF12 polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO:23. In some embodiments, the cells express a TAF12 polypeptide that comprises one or more amino acid substitutions that reduce its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the cells express a TAF12 polypeptide that comprises one or more deletions or insertions that reduce its susceptibility to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 23.

[0381] In some embodiments, the cells express a TAF12 polypeptide that contains one or more amino acid substitutions, or one or more insertions or deletions, that increase the yield of or promote the formation of inclusion bodies compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO: 11.

[0382] In some embodiments, the TAF12 polypeptide comprises at least one amino acid sequence having between 80% and 85%, 85% and 90%, 90% and 95%, or 95% and 100% sequence identity to any one of SEQ ID NOs: 12-14 and 31-2401. In some embodiments, the TAF12 polypeptide comprises a modified N-terminus and / or C-terminus. In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions at the N-terminus and / or C-terminus relative to SEQ ID NO: 23. In some embodiments, the TAF12 polypeptide comprises 11 amino acid substitutions at the N-terminus relative to SEQ ID NO: 23. In some embodiments, the TAF12 polypeptide comprises a sequence tag.

[0383] X.Kit Some aspects of the present disclosure provide kits that include any of the fusion polypeptides, insoluble carrier polypeptides (e.g., onconase polypeptides or TAF12 polypeptides), or oligopeptides of the present disclosure; any of the nucleic acids or vectors encoding the fusion polypeptides, insoluble carrier polypeptides (e.g., onconase polypeptides or TAF12 polypeptides), or oligopeptides of the present disclosure; or cells that express the fusion polypeptides, insoluble carrier polypeptides (e.g., onconase polypeptides or TAF12 polypeptides), or oligopeptides of the present disclosure.

[0384] Also provided herein are kits for use in any of the disclosed methods. [Example]

[0385] The following examples further illustrate the present invention but should not be construed as limiting its scope in any way. In light of the present disclosure and the general level of skill in the art, those skilled in the art will recognize that the following examples are intended to be exemplary only, and that numerous changes, modifications, and variations can be made without departing from the scope of the subject matter of the present disclosure. The accompanying drawings should be considered an integral part of the specification and description of the present disclosure.

[0386] Example 1 Conditions for release of peptides from onconase inclusion bodies. This example describes the evaluation of conditions for cleaving DP bonds and releasing peptides from onconase inclusion bodies for the production of short peptides in E. coli.

[0387] method Peptide release from Onconase The onconase fusion construct (Figure 1A) was expressed in E. coli. Bacterial cells were pelleted, and the cell pellet was then lysed by sonication. The cell lysate was pelleted by centrifugation. The pellet was then washed three times with Triton X-100 buffer (0.1 M Tris, 2% Triton X-100, 2 M urea, 10 mM EDTA, pH 7.4), followed by two washes with wash buffer (1 M Tris, pH 7.4), and then with water. The purified inclusion bodies were then subjected to chemical cleavage by adjusting the target pH with HCl and incubation at 80°C for 16 hours.

[0388] Analysis of peptide expression Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis was used to assess peptide yield and solubilization of onconase (Fig. 1B).

[0389] result In the first experiment, four pH values ​​between 2 and 5 were tested (Figures 2A-2B). While onconase remained mostly in inclusion bodies, chemical cleavage was still reasonably efficient. Cleavage efficiency was most efficient in the pH range of 2 and 3.

[0390] A second experiment was performed to determine the optimal balance between efficient pH cleavage with minimal amounts of solubilized fusion protein. A cleavage pH of 2.4 to 2.8 was the optimal cleavage condition with high levels of released peptide and low amounts of solubilized fusion protein (Figure 3A-B). Similar results were obtained using sulfuric acid to adjust the pH (data not shown).

[0391] To assess the effect on amine groups, model peptides containing glutamine residues were tested. As depicted in Figure 4A-B, when the cleavage pH was lowered below pH 3.0, glutamine-containing peptides began to convert from their basic form to an acidic form, which could be separated by reverse-phase HPLC (Figure 4A). This indicated that glutamine was converted to glutamic acid under acidic conditions, likely through a deamidation reaction catalyzed by high temperature and acidic conditions. This finding emphasizes the importance of operating at a pH above 2.6 for pH-sensitive peptides containing asparagine or glutamine.

[0392] Example 2 Comparison of different inclusion body-forming proteins This example describes the evaluation of different inclusion body-forming proteins for the production of short peptides in E. coli.

[0393] Five different inclusion body-forming proteins were compared: delta(5)-3-ketosteroid isomerase (KSI), the polypeptide F4 fragment derived from the 16.7 kDa PurF protein (PurF); OmpX from Escherichia coli (OmpX), the histone-fold domain of the human transcription factor TAF12 (TAF12), and onconase (Figure 5A). All constructs were cloned into a pET28a plasmid-based expression system, transformed into the BL21(DE3) bacterial strain, and selected on kanamycin (50 μg / ml). Expression experiments were performed in triplicate in flasks at 37°C in autoinduction medium (Figure 5B). The peptide yield of test peptide A was assessed after chemical cleavage (approximately 16 h overnight incubation) at 80°C and pH 2.6 using a synthetic peptide of the same amino acid sequence at a known concentration by RP-HPLC C18 column.

[0394] All five fusion proteins were efficiently expressed and the bioproduced peptides were released under the test conditions.

[0395] Example 3 Concatenated strategies for peptide production in Escherichia coli. This example describes the evaluation of the concatamer strategy for the production of short peptides in E. coli.

[0396] method Peptide production using onconase concatemers The onconase fusion construct was expressed in E. coli. Bacterial cells were pelleted, and the cell pellet was then lysed via sonication. The cell lysate was pelleted by centrifugation. The pellet was then washed three times with Triton X-100 buffer (0.1 M Tris, 2% Triton X-100, 2 M urea, 10 mM EDTA, pH 7.4), followed by two washes with wash buffer (1 M Tris, pH 7.4), and then with water. The purified inclusion bodies were then solubilized by incubation in solubilization buffer (6 M guanidinium chloride, 50 mM Tris, 10 mM beta-mercaptoethanol, pH 7.4). The solubilized protein was then refolded for onconase precipitation by extensive dialysis in 0.1 M acetic acid, pH 3.0, at 4°C, which removed the guanidinium chloride. The soluble fraction was cleaved by incubation in 0.1 M acetic acid at pH 2 and 60°C for 24 hours. After cleavage, onconase was precipitated from the solution by adding NH4OH buffer (pH 7.0-7.2). The peptide was then lyophilized to remove the ammonium acetate, followed by centrifugation.

[0397] Analysis of peptide expression Protein expression was assessed by SDS-PAGE analysis of the insoluble fraction. Expression studies were performed at 37°C in commercial autoinduction medium (Formedium, Ref. AIMTB02) using the BL21(DE3)STAR bacterial strain (Invitrogen, Ref. C601003).

[0398] result The concatemer-onconase fusion strategy could be used to increase the yield of small peptides (e.g., 10-amino acid-long peptides). Concatemer constructs can be designed as homoconcatemers (Figure 6A), in which several copies of the same peptide are separated by acid cleavage sites fused to the C-terminus of onconase, or as heteroconcatemers (Figure 6B), in which different peptide sequences are added.

[0399] To determine whether the concatemer strategy could be applied for efficient peptide production, onconase fusions with three different 10-amino acid-long hydrophilic peptides were prepared. The onconase fusions contained one or three copies of each peptide. As shown in Figure 7, both homo- and heteroconcatemer constructs were expressed at high levels. Addition of up to three copies of each peptide did not affect the accumulation of large amounts of protein in inclusion bodies.

[0400] To verify that the concatemer strategy is applicable to other inclusion body-forming proteins, a fusion TAF12 construct in which three identical peptides were fused to the C-terminus was expressed. The inclusion bodies were washed as in Example 3 and then subjected to cleavage by adjusting the pH to 2.6 with HCl and incubating at 80°C. As shown in Figure 8A, after 4 hours of cleavage, intermediate species corresponding to mono-, di-, and tri-peptide species could be detected. With prolonged cleavage time, almost all were converted to the desired single peptide species (Figure 8B).

[0401] Example 4 N-terminal modifications of onconase for increased peptide yields This example describes the evaluation of mutant onconase proteins to increase peptide yield and purity.

[0402] method Peptide produ...

Claims

1. 1. A method for producing an oligopeptide, comprising: expressing a fusion polypeptide comprising an insoluble carrier polypeptide operably linked by peptide bonds to two or more oligopeptides; and Releasing two or more oligopeptides from an insoluble carrier polypeptide by sequence-specific chemical cleavage of peptide bonds. A method comprising:

2. The method of claim 1, wherein the insoluble carrier polypeptide comprises an onconase polypeptide.

3. The method of claim 1, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

4. Insoluble carrier polypeptides include trpΔLE polypeptide, ketosteroid isomerase (KSI) polypeptide, β-galactosidase polypeptide, PagP polypeptide, truncated Escherichia coli PurF F4 fragment polypeptide, Pseudomonas aeruginosa PaP3.30 polypeptide, histone fold domain of human transcription factor TAF12 (TAF12-HFD) polypeptide, cleavable self-aggregation tag INTEIN-ELK16, Escherichia coli maltose binding protein, Escherichia coli RNAse II polypeptide, Escherichia coli alkaline phosphatase polypeptide, Escherichia coli phospholipase A polypeptide, Escherichia coli β-lactamase polypeptide, Salmonella typhimurium MalK protein, Clostridium thermocellum endoglucanase D polypeptide, Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, human procathepsin B polypeptide, porcine interferon-γ polypeptide, T5 The method of claim 1 , comprising a DNA polymerase polypeptide and an E. coli thioredoxin polypeptide.

5. 5. The method of any one of claims 1 to 4, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

6. 6. The method of any one of claims 1 to 5, wherein two or more oligopeptides are different.

7. The method of any one of claims 1 to 6, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

8. 7. The method of any one of claims 1 to 6, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

9. The method of claim 8, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

10. 10. The method of any one of claims 1 to 9, wherein: (i) the peptide bond comprises methionine and the sequence-specific chemical cleavage uses cyanogen bromide; (ii) the peptide bond comprises tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole; (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid; (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine; or (v) the peptide bond comprises cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

11. 10. The method of claim 1, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage uses acetic acid.

12. 12. The method of any one of claims 1 to 11, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

13. 12. The method of any one of claims 1 to 11, wherein the oligopeptides are operably linked by different peptide bonds and, after the oligopeptides are released from the insoluble carrier polypeptide, are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

14. 14. The method of claim 13, wherein (i) the different peptide bonds comprise methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

15. 14. The method of claim 13, wherein the different peptide bonds comprise Asp-Pro bonds and the sequence-specific chemical cleavage uses acetic acid.

16. 16. The method of any one of claims 1 to 15, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

17. 17. The method of any one of claims 1 to 16, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

18. 16. The method of any one of claims 1 to 15, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

19. 19. The method of any one of claims 1 to 18, wherein the fusion peptide is expressed in bacteria or yeast.

20. 20. The method of claim 19, wherein the bacterium is Escherichia coli or Vibrio natrigens.

21. 21. The method of claim 19 or claim 20, wherein the yield of released oligopeptide is at least 10 mg per liter of bacterial culture, at least 20 mg per liter of bacterial culture, at least 30 mg per liter of bacterial culture, at least 40 mg per liter of bacterial culture, 50 mg per liter of bacterial culture, at least 1 g per liter, or at least 5 g per liter.

22. A fusion polypeptide comprising an insoluble carrier polypeptide operably linked to two or more oligopeptides, wherein the operably linked linkages between the two or more oligopeptides and the insoluble carrier polypeptide comprise peptide bonds capable of sequence-specific chemical cleavage.

23. 23. The fusion polypeptide of claim 22, wherein the insoluble carrier polypeptide comprises an onconase polypeptide.

24. 23. The fusion polypeptide of claim 22, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

25. Insoluble carrier polypeptides include trpΔLE polypeptide, ketosteroid isomerase (KSI) polypeptide, β-galactosidase polypeptide, PagP polypeptide, truncated Escherichia coli PurF F4 fragment polypeptide, Pseudomonas aeruginosa PaP3.30 polypeptide, histone fold domain of human transcription factor TAF12 (TAF12-HFD) polypeptide, cleavable self-aggregation tag INTEIN-ELK16, Escherichia coli maltose binding protein, Escherichia coli RNAse II polypeptide, Escherichia coli alkaline phosphatase polypeptide, Escherichia coli phospholipase A polypeptide, Escherichia coli β-lactamase polypeptide, Salmonella typhimurium MalK protein, Clostridium thermocellum endoglucanase D polypeptide, Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, human procathepsin B polypeptide, porcine interferon-γ polypeptide, T5 23. The fusion polypeptide of claim 22, comprising a DNA polymerase polypeptide and an E. coli thioredoxin polypeptide.

26. 26. The fusion polypeptide of any one of claims 22 to 25, comprising three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

27. 27. The fusion polypeptide of any one of claims 22 to 26, wherein two or more oligopeptides are different.

28. 28. A fusion polypeptide according to any one of claims 22 to 27, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

29. 28. A fusion polypeptide according to any one of claims 22 to 27, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

30. 30. The fusion polypeptide of claim 29, comprising two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

31. 31. The fusion polypeptide of any one of claims 22 to 30, wherein (i) the peptide bond comprises methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

32. 31. The fusion polypeptide of any one of claims 22 to 30, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage uses acetic acid.

33. 33. The fusion polypeptide of any one of claims 22 to 32, wherein the oligopeptides are operably linked by peptide bonds and can be released from each other using sequence-specific chemical cleavage.

34. 33. The fusion polypeptide of any one of claims 22 to 32, wherein the oligopeptides are operably linked by different peptide bonds and can be released from each other by different sequence-specific chemical cleavages.

35. 35. The fusion polypeptide of claim 34, wherein (i) the different peptide bonds comprise methionine and the different sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophan and the different sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the different sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the different sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteine ​​and the different sequence-specific chemical cleavage uses NTCB.

36. 35. The fusion polypeptide of claim 34, wherein the different peptide bonds comprise Asp-Pro bonds and the different sequence-specific chemical cleavage uses acetic acid.

37. 37. The fusion polypeptide of any one of claims 22 to 36, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

38. 38. The fusion polypeptide of any one of claims 22 to 37, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

39. 37. The fusion polypeptide of any one of claims 22 to 36, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

40. 1. A method for releasing an oligopeptide fused to an insoluble carrier polypeptide that forms inclusion bodies in cells, comprising: a) expressing a fusion polypeptide comprising an oligopeptide operably linked to an insoluble carrier polypeptide, wherein the operably linked peptide bond is capable of sequence-specific chemical cleavage with acetic acid; b) purifying the inclusion bodies, and c) incubating the inclusion bodies with acid for at least 1 hour at a temperature greater than 50°C, wherein the oligopeptide is released from the fusion polypeptide by sequence-specific cleavage of peptide bonds. A method comprising:

41. 41. The method of claim 40, wherein the insoluble carrier polypeptide comprises an onconase polypeptide.

42. 41. The method of claim 40, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

43. Insoluble carrier polypeptides include trpΔLE polypeptide, ketosteroid isomerase (KSI) polypeptide, β-galactosidase polypeptide, PagP polypeptide, truncated Escherichia coli PurF F4 fragment polypeptide, Pseudomonas aeruginosa PaP3.30 polypeptide, histone fold domain of human transcription factor TAF12 (TAF12-HFD) polypeptide, cleavable self-aggregation tag INTEIN-ELK16, Escherichia coli maltose binding protein, Escherichia coli RNAse II polypeptide, Escherichia coli alkaline phosphatase polypeptide, Escherichia coli phospholipase A polypeptide, Escherichia coli β-lactamase polypeptide, Salmonella typhimurium MalK protein, Clostridium thermocellum endoglucanase D polypeptide, Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, human procathepsin B polypeptide, porcine interferon-γ polypeptide, T5 41. The method of claim 40, comprising a DNA polymerase polypeptide and an E. coli thioredoxin polypeptide.

44. 44. The method of any one of claims 40 to 43, wherein the temperature in step c) is greater than 60°C, greater than 70°C, greater than 80°C, or greater than 90°C.

45. 45. The method of any one of claims 40 to 44, wherein the temperature in step c) is less than 100°C, less than 95°C, less than 90°C, or less than 85°C.

46. 46. ​​The method of any one of claims 40 to 45, wherein the pH in step c) is below 3.

0.

47. 46. ​​The method according to any one of claims 40 to 45, wherein the pH in step c) is between 2.6 and 2.

8.

48. 48. The method of any one of claims 40 to 47, wherein the acid is a strong acid optionally selected from hydrochloric acid and sulfuric acid.

49. 48. The method of any one of claims 40 to 47, wherein the acid is a weak acid.

50. 50. The method of claim 49, wherein the weak acid is acetic acid.

51. 51. The method of claim 50, wherein the acetic acid concentration is at least 2 weight percent, at least 3 weight percent, at least 4 weight percent, or at least 5 weight percent.

52. 52. The method of claim 50 or claim 51, wherein the acetic acid concentration is less than 50 weight percent, less than 45 weight percent, less than 40 weight percent, less than 35 weight percent, or less than 30 weight percent.

53. 53. The method of any one of claims 40 to 52, wherein the insoluble carrier polypeptide is solubilized by incubation in step c).

54. 53. The method of any one of claims 40 to 52, wherein the insoluble carrier polypeptide is not solubilized by the incubation in step c).

55. 55. The method of any one of claims 40 to 54, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

56. 56. The method of any one of claims 40 to 55, wherein two or more oligopeptides are different.

57. 57. The method of any one of claims 40 to 56, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

58. 57. The method of any one of claims 40 to 56, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

59. 59. The method of claim 58, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

60. 60. The method of any one of claims 40 to 59, wherein (i) the peptide bond comprises methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

61. 61. The method of any one of claims 40 to 60, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is acid cleavage.

62. 62. The method of any one of claims 40 to 61, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

63. 63. The method of any one of claims 40 to 62, wherein the oligopeptides are operably linked by different peptide bonds and, after the oligopeptides are released from the insoluble carrier polypeptide, are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

64. 64. The method of claim 63, wherein (i) the different peptide bonds comprise methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

65. 65. The method of claim 64, wherein the different peptide bonds comprise Asp-Pro bonds and the sequence-specific chemical cleavage is acid cleavage.

66. 66. The method of any one of claims 40 to 65, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

67. 67. The method of any one of claims 40 to 66, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

68. 66. The method of any one of claims 40 to 65, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

69. 69. The method of any one of claims 40 to 68, wherein the fusion peptide is expressed in bacteria or yeast.

70. 70. The method of claim 69, wherein the bacterium is Escherichia coli or Vibrio natrigens.

71. 1. A method for purifying inclusion bodies containing a fusion protein, comprising: a) expressing a fusion protein comprising an oligopeptide operably linked to an insoluble carrier polypeptide that forms inclusion bodies in cells, wherein the operably linkage is a chemically cleavable amino acid sequence; b) lysing the cells to form a cell lysate; c) centrifuging the cell lysate to form a pellet; d) washing the pellet at least once, at least twice, or at least three times with a detergent buffer containing a non-ionic detergent; e) washing the pellet at least once, at least twice, or at least three times with a salt buffer containing at least 0.5 M NaCl; and f) washing the pellet with water at least once, at least twice, or at least three times, thereby producing purified inclusion bodies. A method comprising:

72. 72. The method of claim 71, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

73. 72. The method of claim 71, wherein the insoluble carrier polypeptide comprises an onconase polypeptide.

74. Insoluble carrier polypeptides include trpΔLE polypeptide, ketosteroid isomerase (KSI) polypeptide, β-galactosidase polypeptide, PagP polypeptide, truncated Escherichia coli PurF F4 fragment polypeptide, Pseudomonas aeruginosa PaP3.30 polypeptide, histone fold domain of human transcription factor TAF12 (TAF12-HFD) polypeptide, cleavable self-aggregation tag INTEIN-ELK16, Escherichia coli maltose binding protein, Escherichia coli RNAse II polypeptide, Escherichia coli alkaline phosphatase polypeptide, Escherichia coli phospholipase A polypeptide, Escherichia coli β-lactamase polypeptide, Salmonella typhimurium MalK protein, Clostridium thermocellum endoglucanase D polypeptide, Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, human procathepsin B polypeptide, porcine interferon-γ polypeptide, T5 72. The method of claim 71, comprising a DNA polymerase polypeptide and an E. coli thioredoxin polypeptide.

75. 75. The method of any one of claims 71 to 74, wherein the salt buffer is at least 0.6 M NaCl, at least 0.7 M NaCl, or at least 0.75 M NaCl.

76. 76. The method of any one of claims 71 to 75, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

77. 77. The method of any one of claims 71 to 76, wherein two or more oligopeptides are different.

78. 78. The method of any one of claims 71 to 77, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

79. 78. The method of any one of claims 71 to 77, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

80. 80. The method of claim 79, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

81. 81. The method of any one of claims 71 to 80, wherein (i) the peptide bond comprises methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

82. 81. The method of any one of claims 71 to 80, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is acid cleavage.

83. 83. The method of any one of claims 71 to 82, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

84. 83. The method of any one of claims 71 to 82, wherein the oligopeptides are operably linked by different peptide bonds and, after the oligopeptides are released from the insoluble carrier polypeptide, are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

85. 85. The method of claim 84, wherein (i) the different peptide bonds comprise methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

86. 85. The method of claim 84, wherein the different peptide bonds comprise Asp-Pro bonds and the sequence-specific chemical cleavage uses acetic acid.

87. 87. The method of any one of claims 71 to 86, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

88. 88. The method of any one of claims 71 to 87, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

89. 87. The method of any one of claims 71 to 86, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

90. 90. The method of any one of claims 71 to 89, wherein the fusion peptide is expressed in bacteria or yeast.

91. 91. The method of claim 90, wherein the bacterium is Escherichia coli or Vibrio natrigens.

92. A method for producing a fusion polypeptide, comprising expressing a fusion polypeptide comprising an oligopeptide operably linked to the C-terminus of an onconase polypeptide, wherein the onconase polypeptide comprises one or more amino acid substitutions at the 11 N-terminal amino acids compared to SEQ ID NO: 1, and wherein the onconase-oligopeptide fusion protein comprising the onconase polypeptide is expressed at a higher level than a fusion protein comprising the onconase of SEQ ID NO: 1 when expressed under the same conditions.

93. 93. The method of claim 92, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

94. 94. The method of claim 92 or claim 93, wherein two or more oligopeptides are different.

95. 95. The method of any one of claims 92 to 94, wherein the oligopeptides are all operably linked to the N-terminus of the onconase polypeptide or the oligopeptides are all operably linked to the C-terminus of the onconase polypeptide.

96. 95. The method of any one of claims 92 to 94, wherein at least one oligopeptide is operably linked to the N-terminus of the onconase polypeptide and at least one oligopeptide is operably linked to the C-terminus of the onconase polypeptide.

97. 97. The method of claim 96, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the onconase polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the onconase polypeptide.

98. 98. The method of any one of claims 92 to 97, wherein (i) the peptide bond comprises methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

99. 98. The method of any one of claims 92 to 97, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage uses acetic acid.

100. 100. The method of any one of claims 92 to 99, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from onconase.

101. 100. The method of any one of claims 92 to 99, wherein the oligopeptides are operably linked by different peptide bonds and, after the oligopeptides are released from onconase, are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

102. 102. The method of claim 101, wherein (i) the different peptide bonds comprise methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

103. 102. The method of claim 101, wherein the different peptide bonds comprise Asp-Pro bonds and the sequence-specific chemical cleavage is acid cleavage.

104. 104. The method of any one of claims 92 to 103, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

105. 105. The method of any one of claims 92 to 104, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

106. 104. The method of any one of claims 92 to 103, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

107. 107. The method of any one of claims 92 to 106, wherein the fusion peptide is expressed in bacteria or yeast.

108. 108. The method of claim 107, wherein the bacterium is Escherichia coli or Vibrio natrigens.

109. An onconase polypeptide comprising one or more amino acid substitutions in the 11 N-terminal amino acids of the onconase polypeptide when compared to SEQ ID NO: 1, wherein the onconase polypeptide is expressed at a higher level than the onconase protein of SEQ ID NO: 1 when expressed under the same conditions.

110. 110. The onconase polypeptide of claim 109, comprising the amino acid sequence of one of SEQ ID NOs: 2-10 and 15-22.

111. A fusion polypeptide comprising an onconase polypeptide operably linked to one or more oligopeptides, wherein the operable linkage between the one or more oligopeptides and the onconase polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage, and wherein the onconase polypeptide comprises one or more amino acid substitutions in the 11 N-terminal amino acids of the onconase polypeptide when compared to SEQ ID NO: 1, and wherein the onconase polypeptide is expressed at a higher level than the onconase protein of SEQ ID NO: 1 when expressed under the same conditions.

112. 112. The fusion polypeptide of claim 111, wherein the onconase polypeptide comprises the amino acid sequence of one of SEQ ID NOs: 2-10 and 15-22.

113. 113. The fusion polypeptide of claim 111 or claim 112, comprising three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

114. 114. A fusion polypeptide according to any one of claims 111 to 113, wherein two or more oligopeptides are different.

115. 115. The fusion polypeptide of any one of claims 111 to 114, wherein the oligopeptides are all operably linked to the N-terminus of the onconase polypeptide or the oligopeptides are all operably linked to the C-terminus of the onconase polypeptide.

116. 115. The fusion polypeptide of any one of claims 111 to 114, wherein at least one oligopeptide is operably linked to the N-terminus of the onconase polypeptide and at least one oligopeptide is operably linked to the C-terminus of the onconase polypeptide.

117. The fusion polypeptide of claim 116, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the onconase polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the onconase polypeptide.

118. 118. The fusion polypeptide of any one of claims 111 to 117, wherein (i) the peptide bond comprises methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

119. 118. The fusion polypeptide of any one of claims 111 to 117, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is acid cleavage.

120. 120. The fusion polypeptide of any one of Claims 111 to 119, wherein the oligopeptides are operably linked by peptide bonds and can be released from each other using sequence-specific chemical cleavage.

121. 120. The fusion polypeptide of any one of claims 105 to 119, wherein the oligopeptides are operably linked by different peptide bonds and can be released from each other by different sequence-specific chemical cleavages.

122. 122. The fusion polypeptide of claim 121, wherein (i) the different peptide bonds comprise methionine and the different sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophan and the different sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the different sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the different sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteine ​​and the different sequence-specific chemical cleavage uses NTCB.

123. 122. The fusion polypeptide of claim 121, wherein the different peptide bond comprises an Asp-Pro bond and the different sequence-specific chemical cleavage is acid cleavage.

124. 124. The fusion polypeptide of any one of claims 111 to 123, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

125. 125. The fusion polypeptide of any one of claims 111 to 124, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

126. 126. The fusion polypeptide of any one of claims 111 to 125, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

127. An oligopeptide comprising an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both an N-terminal proline and a C-terminal aspartic acid, wherein the active amino acid sequence is a miPEP and the oligopeptide regulates miRNA; or the active amino acid sequence is a peptide microorganism inhibitor and the oligopeptide inhibits microorganisms.

128. 127. A nucleic acid encoding a fusion polypeptide of any one of claims 22 to 39, an onconase of claim 109 or claim 110, or a fusion polypeptide of any one of claims 111 to 126.

129. A cell comprising the nucleic acid of claim 128.

130. 130. The cell of claim 129, which is a bacterial cell or a yeast cell.

131. 130. The cell of claim 129, wherein the bacterium is Escherichia coli or Vibrio natrigens.

132. 132. The cell of claim 131, which is a BL21 bacterial cell.

133. 132. The cell of claim 131, which does not express Lon and ompT proteases.

134. 129. The nucleic acid of claim 128, which is an isolated nucleic acid.

135. 109. The method of any one of claims 1 to 108, wherein the fusion polypeptide or fusion peptide is expressed in E. coli.

136. 136. The method of any one of claims 1 to 108 and 135, wherein the fusion polypeptide or fusion protein is expressed in cells grown in a fermentation bioreactor.

137. 109. The method of any one of claims 1 to 108, wherein the cleavage is carried out at a pH of about 2 to 3.5 and a temperature of about 70 to 90°C for about 1 to 24 hours.

138. A method for producing a fusion polypeptide, comprising expressing a fusion polypeptide comprising an oligopeptide operably linked to the N-terminus or C-terminus of a modified TAF polypeptide, wherein the modified TAF polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

23.

139. 139. The method of claim 138, wherein the fusion polypeptide comprises three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, or twenty or more oligopeptides.

140. 140. The method of claim 138 or claim 139, wherein two or more oligopeptides are different.

141. 141. The method of any one of claims 138 to 140, wherein the oligopeptides are all operably linked to the N-terminus of the modified TAF polypeptide or the oligopeptides are all operably linked to the C-terminus of the modified TAF polypeptide.

142. 141. The method of any one of claims 138 to 140, wherein at least one oligopeptide is operably linked to the N-terminus of the modified TAF polypeptide and at least one oligopeptide is operably linked to the C-terminus of the modified TAF polypeptide.

143. 143. The method of claim 142, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the modified TAF polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the modified TAF polypeptide.

144. 144. The method of any one of claims 138 to 143, wherein (i) the peptide bond comprises methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

145. 144. The method of any one of claims 138 to 143, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage uses acetic acid or sulfuric acid.

146. 146. The method of any one of claims 138-145, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the modified TAF polypeptide.

147. 146. The method of any one of claims 138 to 145, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the modified TAF polypeptide, they are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

148. 148. The method of claim 147, wherein (i) the different peptide bonds comprise methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the different peptide bonds comprise tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the sequence-specific chemical cleavage uses formic acid, (iv) the different peptide bonds comprise asparagine-glycine bonds and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the different peptide bonds comprise cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

149. 148. The method of claim 147, wherein the different peptide bonds comprise Asp-Pro bonds and the sequence-specific chemical cleavage is acid cleavage.

150. 150. The method of any one of claims 138 to 149, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

151. 151. The method of any one of claims 138 to 150, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

152. 150. The method of any one of claims 138 to 149, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

153. 153. The method of any one of claims 138 to 152, wherein the fusion peptide is expressed in bacteria or yeast.

154. 154. The method of claim 153, wherein the bacterium is Escherichia coli or Vibrio natrigens.

155. A modified TAF polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

23.

156. 156. The modified TAF polypeptide of claim 155, wherein the modified TAF polypeptide comprises the amino acid sequence of SEQ ID NO:

23.

157. A fusion polypeptide comprising a modified TAF polypeptide operably linked to one or more oligopeptides, wherein the operable linkage between the one or more oligopeptides and the modified TAF polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage, and the modified TAF polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

23.

158. 158. The fusion polypeptide of claim 157, wherein the modified TAF polypeptide comprises the amino acid sequence of SEQ ID NO:

23.

159. 159. The fusion polypeptide of claim 157 or claim 158, comprising three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, fifteen or more oligopeptides, twenty or more oligopeptides.

160. 160. The fusion polypeptide of any one of claims 157 to 159, wherein two or more oligopeptides are different.

161. A fusion polypeptide described in any one of claims 157 to 160, wherein all of the oligopeptides are operably linked to the N-terminus of the modified TAF polypeptide or all of the oligopeptides are operably linked to the C-terminus of the modified TAF polypeptide.

162. A fusion polypeptide described in any one of claims 157 to 160, wherein at least one oligopeptide is operably linked to the N-terminus of the modified TAF polypeptide and at least one oligopeptide is operably linked to the C-terminus of the modified TAF polypeptide.

163. 163. The fusion polypeptide of claim 162, comprising two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the modified TAF polypeptide and / or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the modified TAF polypeptide.

164. 164. The fusion polypeptide of any one of claims 157 to 163, wherein (i) the peptide bond comprises a methionine and the sequence-specific chemical cleavage uses cyanogen bromide, (ii) the peptide bond comprises a tryptophan and the sequence-specific chemical cleavage uses BNPS-skatole, (iii) the peptide bond comprises an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage uses formic acid, (iv) the peptide bond comprises an asparagine-glycine bond and the sequence-specific chemical cleavage uses hydroxylamine, or (v) the peptide bond comprises a cysteine ​​and the sequence-specific chemical cleavage uses NTCB.

165. 164. The fusion polypeptide of any one of claims 157 to 163, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is acid cleavage.

166. 166. The fusion polypeptide of any one of Claims 157 to 165, wherein the oligopeptides are operably linked by peptide bonds and can be released from each other using sequence-specific chemical cleavage.

167. 166. The fusion polypeptide of any one of claims 157 to 165, wherein the oligopeptides are operably linked by different peptide bonds and can be released from each other by different sequence-specific chemical cleavages.

168. 168. The fusion polypeptide of claim 167, wherein (i) the different peptide bonds comprise methionine and the different sequence-specific chemical cleavage uses cyanogen bromide; (ii) the different peptide bonds comprise tryptophan and the different sequence-specific chemical cleavage uses BNPS-skatole; (iii) the different peptide bonds comprise aspartic acid-proline (Asp-Pro) bonds and the different sequence-specific chemical cleavage uses formic acid; (iv) the different peptide bonds comprise asparagine-glycine bonds and the different sequence-specific chemical cleavage uses hydroxylamine; or (v) the different peptide bonds comprise cysteine ​​and the different sequence-specific chemical cleavage uses NTCB.

169. 168. The fusion polypeptide of claim 167, wherein the different peptide bond comprises an Asp-Pro bond and the different sequence-specific chemical cleavage is an acid cleavage.

170. 170. The fusion polypeptide of any one of claims 157 to 169, wherein the oligopeptide is at least 4 amino acids in length, at least 5 amino acids in length, at least 6 amino acids in length, at least 7 amino acids in length, at least 8 amino acids in length, at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, or at least 25 amino acids in length.

171. 171. The fusion polypeptide of any one of claims 157 to 170, wherein the oligopeptide is less than 50 amino acids in length, less than 45 amino acids in length, less than 40 amino acids in length, less than 35 amino acids in length, less than 30 amino acids in length, less than 25 amino acids in length, or less than 20 amino acids in length.

172. 172. The fusion polypeptide of any one of claims 157 to 171, wherein the oligopeptide is between 4 and 50 amino acids in length, between 6 and 40 amino acids in length, between 6 and 30 amino acids in length, or between 8 and 25 amino acids in length.

173. 172. A nucleic acid encoding a fusion polypeptide of any one of claims 22 to 39, a modified TAF polypeptide of claim 155 or claim 156, or a fusion polypeptide of any one of claims 157 to 172.

174. A cell comprising the nucleic acid of claim 173.

175. 175. The cell of claim 174, which is a bacterial cell or a yeast cell.

176. 175. The cell of claim 174, wherein the bacterium is Escherichia coli or Vibrio natrigens.

177. 177. The cell of claim 176, which is a BL21 bacterial cell.

178. 177. The cell of claim 176, which does not express Lon and ompT proteases.

179. 174. The nucleic acid of claim 173, which is an isolated nucleic acid.

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