Fusion polypeptides and binding peptides and methods for producing and using same
AssigneeApplied Biomedical Science Institute
InventorsVaughn SMIDER, Ruiqi HUANG, Duncan MCGREGOR, Alexandra STAMBAUGH
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Abstract
Provided herein are fusion polypeptides in which a small polypeptide is modified by fusion with N- and C-termini sequences that interact with one another when in proximity. In some embodiments, the small polypeptide is a cysteine motif binding peptide or a cytokine. Also provided herein are cysteine motif binding peptides, such as from the knob region of an ultralong CDR3, and methods of producing and using the same. Also provided herein are compositions comprising the binding peptides.
Claims163
A modified fusion polypeptide comprising the formula N-terminus to C-terminus:
- Y1-[polypeptide]-Y2, wherein the polypeptide is a protein in which the distance between the N-termini and C-termini is no more than 10 Angstroms and wherein Y1 and Y2 are amino acid peptide sequences that interact with one another when in proximity.
A method of producing a modified fusion polypeptide, the method comprising adding an N-terminus sequence (Y1) and a C-terminus sequence (Y2) to a polypeptide, wherein the polypeptide is a protein in which the distance between the N-termini and C-termini is no more than 10 Angstroms and wherein the modified fusion polypeptide has the formula N-terminus to C-terminus:
- Y1-[polypeptide]-Y2, wherein Y1 and Y2 are amino acid peptide sequences that interact with one another when in proximity.
A method of producing a modified binding peptide, the method comprising:
- (a) obtaining a cysteine motif binding peptide of 20-50 amino acids in length in which 2-12 amino acids are cysteine residues able to form 1-6 disulfide bonds;
- and
- (b) modifying the binding peptide by adding an N-terminus sequence (Y1) and a C-terminus sequence (Y2) to the cysteine motif binding peptide, wherein the modified binding peptide has the formula N-terminus to C-terminus:
- Y1-[cysteine motif binding peptide]-Y2, wherein Y1 and Y2 are characterized by:
- (i) Y1 and Y2 are selected from (i) HW and SF, respectively;
- (ii) IS and TV, respectively;
- (iii) DY and MP, respectively;
- (iv) LV and IP, respectively;
- or (v) SV and YI, respectively;
- or
- (ii) Y1 and Y2 are sequences that are each 14-30 amino acids in length comprising a heptad repeat, wherein Y1 and Y2 are able to interact to form an anti-parallel coiled-coil motif.
A method for producing a soluble binding peptide, comprising:
- (a) transforming E. coli with an expression vector encoding a fusion protein comprising a binding peptide and thioredoxin A (TrxA) bacterial chaperone set forth in SEQ ID NO:194 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:194, wherein the binding peptide is a cysteine modified binding peptide of 20-50 amino acids in length in which 2-12 amino acids are cysteine residues able to form 1-6 disulfide bonds;
- and;
- (b) culturing the bacteria under conditions permissive of expression of the fusion protein;
- and
- (c) isolating the fusion protein from supernatant of a bacterial cell lysate.
A method for producing a soluble ultralong CDR3 knob, comprising:
- (a) transforming E. coli with an expression vector encoding a fusion protein comprising a binding peptide and a bacterial chaperone, wherein the binding peptide is a modified binding peptide that has the formula N-terminus to C-terminus:
- Y1-[cysteine motif binding peptide]-Y2, wherein the cysteine motif binding peptide is a peptide sequence of 20-50 amino acids with a cysteine motif comprising 2-12 cysteine residues able to form 1-6 disulfide bonds and Y1 and Y2 are characterized by:
- (i) Y1 and Y2 are selected from (i) HW and SF, respectively;
- (ii) IS and TV, respectively;
- (iii) DY and MP, respectively;
- (iv) LV and IP, respectively;
- or (v) SV and YI, respectively;
- or
- (ii) Y1 and Y2 are sequences that are each 14-30 amino acids in length comprising a heptad repeat, wherein Y1 and Y2 are able to interact to form an anti-parallel coiled-coil motif;
- (b) culturing the bacteria under conditions permissive of expression of the fusion protein;
- and
- (c) isolating the fusion protein from supernatant of a bacterial cell lysate.
A method for producing a soluble binding peptide, comprising:
- (a) transforming E. coli with an expression vector encoding a fusion protein comprising a binding peptide and a bacterial chaperone, wherein the binding peptide is set forth in any one of SEQ ID NOS:
- 60, 61, 62, 63, 64, 65, 66, 67, 68, 155, 198 and 227-317 and;
- (b) culturing the bacteria under conditions permissive of expression of the fusion protein;
- (c) isolating the fusion protein from supernatant of a bacterial cell lysate.
A fusion protein comprising a modified binding peptide and a bacterial chaperone joined by a cleavable linker, wherein the modified binding peptide has the formula N-terminus to C-terminus:
- Y1-[cysteine motif binding peptide]-Y2, wherein:
- the cysteine motif binding peptide is 20-50 amino acids in length in which 2-12 amino acids are cysteine residues able to form 1-6 disulfide bonds;
- and
- Y1 and Y2 are amino acid peptide sequences that interact with one another when in proximity.
A soluble peptide comprising a modified binding peptide that is disulfide-bonded, wherein the modified binding peptide has the formula N-terminus to C-terminus:
- Y1-[cysteine motif binding peptide]-Y2, wherein:
- the cysteine motif binding peptide is 20-50 amino acids in length in which 2-12 amino acids are cysteine residues and wherein the soluble peptide contains 1 to 6 disulfide bonds;
- and
- Y1 and Y2 are amino acid peptide sequences that interact with one another when in proximity.
Dependencies are inferred from the claim text.