Polypeptide display libraries and methods of making and using thereof

• US 20050196406A1
• Filed: 08/18/2004
• Published: 09/08/2005
• Est. Priority Date: 08/18/2003
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Rice, Jeffrey, Daugherty, Patrick S., Bessette, Paul H.
• Granted Patent: US 7,256,038 B2
• Time in Patent Office: Days
• US Class Current: 424/190.1
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 33/554 the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells

Abstract

Disclosed herein are expression vectors which display a passenger polypeptide on the outer surface of a biological entity. As disclosed herein the displayed passenger polypeptide is capable of interacting or binding with a given ligand. Also disclosed are methods of making and using the expression vectors. N/C terminal fusion expression vectors and methods of making and using are also disclosed.

Claims

1. An expression vector capable of expressing and displaying a given passenger polypeptide on an outer surface of a biological entity within a carrier protein loop that is capable of interacting with a given ligand.

2. The expression vector of claim 1, wherein the carrier protein loop is opened resulting in an N-terminus exposed on the outer surface, a C-terminus exposed on the outer surface, or both.

3. The expression vector of claim 2, wherein the native C-terminus and the native N-terminus are fused together via a peptide linker.

4. The expression vector of claim 2, wherein the N-terminus and the C-terminus exposed to the outer surface are accessible by the ligand.

5. The expression vector of claim 2, wherein the C terminus of the passenger polypeptide is fused to the N terminus of the carrier protein.

6. The expression vector of claim 2, wherein the N terminus of the passenger polypeptide is fused to the C terminus of the carrier protein.

7. The expression vector of claim 1, wherein the carrier protein is a bacterial outer membrane protein.

8. The expression vector of claim 1, wherein the bacterial outer membrane protein is OmpA or OmpX.

9. The expression vector of claim 8, wherein the polypeptide is expressed in the first extracellular loop of OmpA.

10. The expression vector of claim 8, wherein the polypeptide is expressed in the second extracellular loop of OmpX.

11. The expression vector of claim 8, wherein the polypeptide is expressed in the third extracellular loop of OmpX.

12. The expression vector of claim 2, wherein the carrier protein is OmpX.

13. The expression vector of claim 1, wherein the polypeptide is streptavidin or a T7 binding peptide.

14. The expression vector of claim 1, wherein the biological entity is a bacterial cell, a yeast cell or a mammalian cell.

15. The expression vector of claim 1, wherein the biological entity is a bacterial cell.

16. The expression vector of claim 15, wherein the bacterial cell is Escherichia coli, Shigella sonnei, Shigella dysenteriae, Shingella flexneri, Salmonella typhimurium, Salmonella enterica, Enterobacter aerogenes, Serratia marcescens, Yersinia pestis, or Klebsiella pneumoniae.

17. The expression vector of claim 1, and further comprising a low copy origin of replication.

18. The expression vector of claim 17, wherein the low copy origin of replication is a p15A origin of replication.

19. The expression vector of claim 1, and further comprising a bacteriocidal antibiotic resistance protein encoding gene.

20. The expression vector of claim 19, wherein the bacteriocidal antibiotic resistant protein encoding gene encodes chloramphenicol acetlytransferase.

21. The expression vector of claim 1, and further comprising at least one SfiI endonuclease restriction enzyme site.

22. The expression vector of claim 1, and further comprising an arabinose araBAD E. coli operon promoter.

23. The expression vector of claim 22, wherein expression is induced with the addition of L-arabinose and stopped by the removal of arabinose and the addition of glucose.

24. A host cell comprising the expression vector of claim 1.

25. A method of making a polypeptide display library which comprises creating a plurality of expression vectors capable of expressing a plurality of polypeptides according to claim 1 and inducing expression.

26. A polypeptide expressed on the outer surface of a biological entity by inducing expression of the expression vector of claim 1.

27. The polypeptide of claim 26, wherein the polypeptide is expressed in the first extracellular loop of OmpA.

28. The polypeptide of claim 26, wherein the polypeptide is expressed in the second extracellular loop of OmpX.

29. The polypeptide of claim 26, wherein the polypeptide is expressed in the third extracellular loop of OmpX.

30. A polypeptide expressed on the outer surface of a biological entity by inducing expression of the expression vector of claim 2.

31. The polypeptide of claim 30, wherein the polypeptide is expressed in OmpX.

32. A polypeptide display library which comprises a polypeptide expressed and displayed by the expression vector of claim 1.

33. A polypeptide display library which comprises a polypeptide expressed and displayed by the expression vector of claim 2.

34. An assay method for detecting, monitoring, or measuring a given ligand in a sample which comprises inducing the expression vector of claim 1 to express the polypeptide and then contacting the polypeptide with the sample and observing whether the polypeptide interacts with the ligand.

35. An assay method for detecting, monitoring, or measuring a given ligand in a sample which comprises inducing the expression vector of claim 2 to express the polypeptide and then contacting the polypeptide with the sample and observing whether the polypeptide interacts with the ligand.

36. The expression vector of claim 1, wherein the carrier polypeptide is encoded by a nucleic acid molecule which comprises at least one codon that encodes a given amino acid that is replaced with a replacement codon which encodes an alternate amino acid that is structurally similar to the given amino acid.

37. The expression vector of claim 36, wherein all the codons that encode the given amino acid are replaced.

38. The expression vector of claim 36, wherein the biological entity incorporates at least one non-canonical amino acid analog into the displayed polypeptide.

39. The expression vector of claim 36, wherein the given amino acid is leucine.

40. The expression vector of claim 39, wherein the alternate amino acid is valine, isoleucine, or trifluorleucine.