Directed evolution of disulfide-bonded micro-proteins
View Patent ↗The invention relates, in part, to a library of chimeric proteins, each chimeric protein including a mini-protein between about eight and about forty amino acids long, wherein the mini-protein has a single disulfide bond formed by a pair of invariant cysteines and has only two cysteines. The chimeric protein also includes at least a portion of an outer surface protein of a genetic package, wherein the chimeric protein is displayed on the outer surface of the genetic package. The invention also includes, in part, a mixture of nucleic acids that encode a library of the invention. The invention also includes, in part, a process for identifying proteins with a desired binding activity against a target, the process including screening a library of chimeric proteins of the invention; and identifying the chimeric protein. The invention, in part, also includes chimeric proteins expressed by a library of the invention.
1. A library of chimeric proteins, each chimeric protein comprising;
a) a mini-protein between about eight and about forty amino acids long, wherein the mini-protein has a single disulfide bond formed by a pair of invariant cysteines, wherein there are no cysteines between the invariant cysteines, and
b) at least a portion of an outer surface protein of a genetic package,
wherein the chimeric protein is displayed on the outer surface of the genetic package.
2. The library of claim 1 , wherein the span of the disulfide bond is at least two amino acid residues and no more than about nine amino acid residues.
3. The library of claim 1 , wherein the span of the disulfide bond is 3 amino acids.
4. The library of claim 1 , wherein the span of the disulfide bond is 4 amino acids.
5. The library of claim 1 , wherein the span of the disulfide bond is 5 amino acids.
6. The library of claim 1 , wherein the span of the disulfide bond is 6 amino acids.
7. The library of claim 1 , wherein the span of the disulfide bond is 7-9 amino acids.
8. The library of claim 1 , wherein at least one of the amino acids in the span of the disulfide bond is Gly in at least some of the mini-proteins in the library.
9. The library of claim 1 , wherein the span of the disulfide bond is 2 or 3 and wherein one of the amino acids in the span of the disulfide bond is Gly in essentially all of the mini-proteins in the library.
10. The library of claim 1 , wherein
the spacing of the disulfide bond is six and
wherein the span of the disulfide bond comprises at least one main-chain geometry constraining amino acid, in each case independently selected from the group consisting of proline, valine, and isoleucine,
in at least some of the mini-proteins in the library.
11. The library of claim 10 , wherein the at least one main-chain geometry constraining amino acid is proline.
12. The library of claim 1 , wherein
the spacing of the disulfide bond is six and
wherein the span of the disulfide bond comprises at least one main-chain geometry constraining amino acid, in each case independently selected from the group consisting of proline, valine, and isoleucine,
in essentially all of the mini-proteins in the library.
13. The library of claim 12 , wherein the main-chain geometry constraining amino acid is proline.
14. The library of claim 1 , wherein the mini-protein comprises an amino acid sequence of 20 or fewer amino acids.
15. The library of claim 1 , wherein each amino acid within the span can be one of at least eight different amino acids.
16. The library of claim 1 , wherein all amino acids included in the span of the disulfide bond, other than any main-chain geometry constraining amino acids, are variable.
17. The library of claim 1 , where the mini-protein comprises the sequence
X 1 -C-X 2 -X 3 -X 4 -X 5 -C-X 6
wherein the cysteines (Cs) form a disulfide bond, and wherein X 2 to X 5 are any amino acids except for cysteine.
18. The library of claim 1 wherein the disulfide bond bridges a sequence of amino acids that collectively assume a hairpin secondary structure under affinity separation conditions.
19. The library of claim 18 , wherein the hairpin secondary structure is a) an alpha helix, a turn, and a beta strand; b) an alpha helix, a turn, and an alpha helix; or c) a beta strand, a turn, and a beta strand.
20. The library of claim 1 , wherein at least one amino acid in the span of the disulfide bond is an amino acid that can be a variegated codon chosen from NNG, RNG, RMG, VNT, RRS, NHT, VNS, and SNT.
21. The library of claim 1 , wherein none of the amino acids in the span of the disulfide bond is an amino acid that can be encoded by a variegated codon chosen from NNN, NNK and NNS.
22. The library of claim 1 , wherein the mini-protein is directly fused to at least a portion of the outer surface protein.
23. The library of claim 1 , wherein the genetic package is a filamentous phage.
24. The library of claim 23 , wherein the mini-protein is encoded by a phagemid.
25. The library of claim 23 , wherein the outer surface protein is the major coat protein of a filamentaus phage.
26. The library of claim 23 , wherein the outer surface protein is the gene III protein of a filamentous phage.
27. The library of claim 1 , wherein the genetic package is a bacterial cell.
28. The library of claim 27 , wherein the bacterial cell is Salmonella typhimuriurn, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis , or Escherichia coli.
29. The library of claim 1 , wherein the genetic package is a bacterial spore.
30. The library of claim 1 , wherein the chimeric protein binds a target.
31. The library of claim 30 , wherein the target is a serine protease.
32. The library of claim 31 , wherein the serine protease is human neutrophil elastase.
33. A process for identifying proteins with a desired binding activity against a target which comprises:
a) screening a library of chimeric proteins according to claim 1 ; and
b) identifying the chimeric protein.
34. The library of claim 1 , wherein the mini-protein further comprises two amino acids that precede the first invariant cysteine and two amino acids that follow the second invariant cysteine in the amino acid sequence of the mini-protein.