Directed evolution of disulfide-bonded micro-proteins

• US 7,413,537 B2
• Filed: 07/31/2002
• Issued: 08/19/2008
• Est. Priority Date: 09/01/1989
• Status: Expired due to Term

• Current Assignee: Dyax Corporation (Shire plc)
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ladner, Robert Charles, Ley, Arthur Charles, Kent, Rachel Baribault, Markland, William, Roberts, Bruce Lindsay, Guterman, Sonia Kosow
• Primary Examiner(s): Schultz; J. Douglas
• Assistant Examiner(s): Lundgren; Jeffrey S.
• Application Number:
  US10/207,797
• Publication Number:
  US 20060084113A1
• Time in Patent Office: 2,211 Days
• Field of Search: None
• US Class Current: 506/14
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

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.

Claims

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

X₁-C-X₂-X₃-X₄-X₅-C-X₆

wherein the cysteines (Cs) form a disulfide bond, and wherein X₂ to X₅ 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.