Methods of producing high diversity peptide libraries and promoting protein folding

A library comprising a plurality of peptides, wherein a peptide of the plurality of peptides comprises:

• a) at least one N-terminus amino acid residue of the peptide;
• b) a cleavable moiety;
• and
• c) a remainder of the peptide, wherein the at least one N-terminus amino acid residue of the peptide is before or within the cleavable moiety;
• wherein the plurality of peptides comprises greater than 1000 peptide diversity when the cleavable moiety is cleaved using an endoprotease specific to the cleaveable moiety, thereby cleaving the at least one N-terminus amino acid residue of the peptide.

A method of making a peptide library, the method comprising:

• a) providing a plurality of nucleic acid constructs encoding a plurality of peptides, wherein a nucleic acid construct of the plurality of nucleic acid constructs comprises:
• i) a first nucleotide sequence encoding a peptide from the plurality of peptides;
• and
• ii) a second nucleotide sequence encoding a cleavable moiety, wherein the cleavable moiety is situated such that at least one N-terminus amino acid residue of the peptide selected from the plurality of peptides is before or within the cleavable moiety;
• b) transcribing and translating, or translating, the plurality of nucleic acid constructs;
• and
• c) cleaving the cleavable moiety using an endoprotease, optionally, simultaneously as (b), thereby cleaving the at least one N-terminus amino acid residue of the peptide from the remainder of the peptide,
• wherein cleavage of the at least one N-terminus amino acid residue from the peptide results in a properly folded peptide of the peptide library.

A DNA construct for expression of a protein epitope, the DNA construct comprising:

• a) a first nucleotide sequence encoding the protein epitope;
• and
• b) a second nucleotide sequence encoding a cleavable moiety at the N-terminus of the protein epitope, wherein the cleavable moiety is situated such that at least one N-terminus amino acid residue of the protein epitope is before or within the cleavable moiety, wherein upon transcription and translation of the DNA construct, the cleavable moiety is cleaved using an endoprotease specific to the cleavable moiety, thereby cleaving the at least one N-terminus amino acid residue of the protein epitope, and
• wherein the protein epitope is part of a peptide library.

A RNA construct for expression of a protein epitope, the RNA construct comprising:

• a) a first nucleotide sequence encoding the protein epitope;
• and
• b) a second nucleotide sequence encoding a cleavable moiety at the N-terminus of the protein epitope, wherein the cleavable moiety is situated such that at least one N-terminus amino acid residue of the protein epitope is before or within the cleavable moiety, wherein upon translation of the RNA construct, the cleavable moiety is cleaved using an endoprotease specific to the cleavable moiety, thereby cleaving the at least one N-terminus amino acid residue of the protein epitope, and
• wherein the protein epitope is part of a peptide library.

A method of folding a peptide, the method comprising:

• a) providing a nucleic acid construct encoding the peptide, the nucleic acid construct comprising:
• i) a first nucleotide sequence encoding the peptide;
• and
• ii) a second nucleotide sequence encoding a cleavable moiety, wherein the cleavable moiety is situated such that at least one N-terminus amino acid residue of the peptide is before or within the cleavable moiety;
• b) transcribing and translating, or translating, the nucleic acid construct;
• and
• c) cleaving the cleavable moiety using an endoprotease, optionally, simultaneously as (b), thereby cleaving the at least one N-terminus amino acid residue of the peptide from the remainder of the peptide,
• wherein cleavage of the at least one N-terminus amino acid residue of the peptide results in a folded peptide.

A method for making a library of conformational protein epitopes, the method comprising:

• a) obtaining a plurality of protein epitopes encoded in a plurality of nucleic acid constructs, wherein a nucleic acid construct of the plurality further encodes a cleavable moiety at the N-terminus of a protein epitope, wherein the cleavage moiety is situated such that an initial amino acid residue of the protein epitope is before or within the cleavable moiety;
• b) optionally transcribing the plurality of nucleic acid constructs, wherein a plurality of ribonucleic acid molecules is transcribed from the plurality of nucleic acid constructs;
• c) translating the plurality of ribonucleic acid molecules, wherein the plurality of protein epitopes are translated from the plurality of ribonucleic acid molecules;
• and
• d) cleaving the cleavable moiety using a protease, optionally, simultaneously as (c), thereby cleaving the initial amino acid residue of the candidate protein epitopes from the remainder of the candidate protein epitopes.