Methods for enhancing bacterial cell display of proteins and peptides

• US 20090062142A1
• Filed: 07/24/2008
• Published: 03/05/2009
• Est. Priority Date: 07/26/2007
• 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.
• Granted Patent: US 8,293,685 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Abstract

Methods of making and using bacterial display polypeptide libraries using circularly permuted OmpX (CPX) variants are disclosed. The invention further relates to methods for enhancing the display of proteins and peptides at the surface of bacteria by optimizing linkers and incorporating mutations at positions 165 and 166 of CPX.

Claims

1. A circularly permuted OmpX (CPX) variant comprising a linker joining the native N-terminus and C-terminus, wherein the linker is 3-8 residues in length and comprises a glycine and one or more basic amino acids.

2. The CPX variant of claim 1, wherein the first residue of the linker is a glycine.

3. The CPX variant of claim 1, wherein the linker comprises at least two basic residues.

4. The CPX variant of claim 1, wherein the linker comprises two arginine residues.

5. The CPX variant of claim 1, wherein the linker comprises two lysine residues.

6. The CPX variant of claim 1, wherein the linker comprises at least one arginine residue and at least one lysine residue.

7. The CPX variant of claim 1, wherein the linker is 5 residues in length.

8. The CPX variant of claim 1, wherein the linker is 6 residues in length.

9. The CPX variant of claim 8, wherein the first residue of the linker is a glycine and the third and sixth residues of the linker are selected from the group consisting of arginine, lysine, serine, histidine, glutamine, and asparagine.

10. The CPX variant of claim 1, wherein the linker comprises a sequence selected from the group consisting of SEQ ID NOS:

2-27.

11. The CPX variant of claim 1, further comprising one or more mutations that increase the display efficiency of a passenger peptide compared to the CPX variant in the absence of the mutations, wherein at least one mutation is at a position corresponding to A165 or G166 of the native OmpX protein numbered relative to the reference sequence of SEQ ID NO:

1.

12. The CPX variant of claim 11, comprising one or more mutations selected from the group consisting of an A165V mutation, an A165L mutation, an A1651 mutation, an A165F mutation, a G166S mutation, a G166A mutation and combinations thereof.

13. The CPX variant of claim 1, further comprising a passenger polypeptide fused to the N-terminus.

14. The CPX variant of claim 13, further comprising a linker between the N-terminus and the passenger polypeptide, wherein said linker comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

15. The CPX variant of claim 1, further comprising a passenger polypeptide fused to the C-terminus.

16. The CPX variant of claim 15, further comprising a linker between the C-terminus and the passenger polypeptide, wherein said linker comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

17. The CPX variant of claim 1, further comprising a first passenger polypeptide fused to the N-terminus and a second passenger polypeptide fused to the C-terminus.

18. The CPX variant of claim 17, wherein the first passenger polypeptide or the second passenger polypeptide further comprises a detectable label.

19. The CPX variant of claim 18, wherein the first passenger polypeptide or the second passenger polypeptide comprises a streptavidin binding peptide.

20. The CPX variant of claim 19, wherein the detectable label is streptavidin conjugated to a fluorophore.

21. The CPX variant of claim 19, wherein the streptavidin binding peptide comprises the sequence of SEQ ID NO:

36.

22. The CPX variant of claim 18, wherein both the first passenger polypeptide and the second passenger polypeptide comprise detectable labels.

23. The CPX variant of claim 22, wherein the first passenger polypeptide comprises a different detectable label than the second passenger polypeptide.

24. The CPX variant of claim 17, further comprising a linker between the first passenger polypeptide and the N-terminus or the second passenger polypeptide and the C-terminus.

25. The CPX variant of claim 24, wherein the linker comprises a sequence selected from the group consisting of SEQ ID NO:

28 and SEQ ID NO;

34.

26. A polynucleotide encoding the CPX variant of claim 1.

27. An expression vector comprising the polynucleotide of claim 26 operably linked to a promoter, wherein the expressed CPX variant displays one or more passenger polypeptides on an outer surface of a bacterial cell.

28. A bacterial cell comprising the expression vector of claim 27.

29. The bacterial cell of claim 28, where 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.

30. A polypeptide display library comprising a polypeptide displayed by the CPX variant of claim 1.

31. A method of making the polypeptide display library of claim 30, the method comprising:

providing a plurality of expression vectors expressing CPX variants carrying a plurality of passenger polypeptides,

transfecting bacterial cells with said expression vectors, and

culturing the bacterial cells under conditions that permit expression of said passenger polypeptides on the surface of the bacterial cells.

32. A method of screening for a CPX variant that displays a passenger polypeptide with greater efficiency than another carrier protein carrying the same passenger polypeptide, the method comprising:

transfecting a bacterial cell with an expression vector expressing a CPX variant carrying the passenger polypeptide,

screening for display of the passenger polypeptide at the surface of the bacterial cell within 25 minutes after inducing the expression of the CPX variant carrying the passenger polypeptide; and

comparing the display efficiency of the CPX variant carrying the passenger polypeptide to the display efficiency of another carrier protein carrying the same passenger polypeptide expressed under the same conditions.

33. A method of enhancing the display efficiency of a passenger polypeptide, the method comprising:

screening a plurality of different CPX variants carrying the same passenger polypeptide according to the method of claim 32, wherein each CPX variant comprises a different linker joining the native N-terminus and C-terminus, wherein the linker is 3-8 residues in length and comprises a glycine and one or more basic amino acids, and optionally, a mutation at a position corresponding to A165 or G166 of the native OmpX protein; and

selecting the CPX variant that displays the passenger polypeptide with the greatest efficiency compared to the plurality of other CPX variants.

34. The method of claim 33, wherein the first residue of the linker is a glycine and the third residue of the linker is selected from the group consisting of arginine, lysine, serine, histidine, glutamine, and asparagine.

35. The method of claim 34, wherein the linker is 6 residues in length.

36. The method of claim 35, wherein the sixth residue of the linker is selected from the group consisting of arginine, lysine, serine, histidine, glutamine, and asparagine.

37. A method of screening a library of polypeptides for the ability to bind to a target molecule, the method comprising:

a) providing a polypeptide display library comprising CPX variants carrying a plurality of passenger polypeptides displayed on bacterial cells,

b) contacting the plurality of passenger polypeptides with the target molecule, and

c) identifying at least one displayed passenger polypeptide that binds to the target molecule.

38. The method of claim 37, wherein the target molecule is selected from the group consisting of a receptor, a ligand, an antibody, an antigen, an enzyme, a transporter, a substrate, an inhibitor, an activator, a cofactor, a drug, a nucleic acid, a lipid, a carbohydrate, a glycoprotein, a small organic molecule, and an inorganic molecule.

39. The method of claim 37, wherein said target molecule comprises a detectable label, wherein identifying the target molecule bound to at least one passenger polypeptide comprises detecting the label attached to said target molecule.

40. A method of screening a library of polypeptides for biological activity in the presence of a target molecule, the method comprising:

a) providing a polypeptide display library comprising CPX variants carrying a plurality of passenger polypeptides displayed on bacterial cells,

b) contacting the plurality of passenger polypeptides with the target molecule,

c) assaying for biological activity in the presence of the target molecule, and

d) identifying at least one displayed passenger polypeptide that has biological activity.

41. The method of claim 40, wherein the biological activity is enzymatic activity, substrate activity, ligand-binding activity, transport activity, agonist activity, or antagonist activity.

42. The method of claim 40, wherein the target molecule is selected from the group consisting of a receptor, a ligand, an antibody, an antigen, an enzyme, a transporter, a substrate, an inhibitor, an activator, a cofactor, a drug, a nucleic acid, a lipid, a carbohydrate, a glycoprotein, a small organic molecule, and an inorganic molecule.