Combinatorial protein library screening by periplasmic expression

• US 20060029947A1
• Filed: 03/18/2005
• Published: 02/09/2006
• Est. Priority Date: 03/18/2004
• Status: Abandoned Application

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Iverson, Brent L., Georgiou, George, Jeong, Ki Jun
• Application Number:
  US11/084,055
• Publication Number:
  US 20060029947A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/034 containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
• CPC Class Codes: C07K 2319/21 containing a His-tag
• 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 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• 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
• CPC Class Codes: G01N 33/5082 Supracellular entities, e.g. tissue, organisms
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides. In the technique, libraries of candidate binding proteins, such as antibody sequences, may be expressed in the periplasm of gram negative bacteria with at least one target ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the ligand becomes bound and retained by the cell even after removal of the outer membrane, allowing the cell to be isolated from cells not expressing a binding polypeptide with affinity for the target ligand. The target ligand may be detected in numerous ways, including use of direct fluorescence or secondary antibodies that are fluorescently labeled, allowing use of efficient screening techniques such as fluorescence activated cell sorting (FACS). The approach is more rapid and robust than prior art methods and avoids problems associated with the outer surface-expression of ligand fusion proteins employed with phage display.

Claims

1. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:

(a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm;

said bacterium comprising a nucleic acid sequence encoding a candidate binding polypeptide comprising an inner membrane anchor polypeptide;

wherein the bacterium further comprises a nucleic acid sequence encoding a target ligand and wherein the target ligand is exported to the periplasm;

(b) allowing the target ligand to bind to the candidate binding polypeptide in said periplasm;

(c) removing unbound target ligand from said periplasm; and

(d) selecting the bacterium based on the presence of the target ligand bound to the candidate binding polypeptide.

2. The method of claim 1, further defined as a method of obtaining a nucleic acid sequence encoding a binding polypeptide having a specific affinity for a target ligand, the method further comprising the step of:

(d) cloning said nucleic acid sequence encoding a candidate binding polypeptide from said bacterium.

3. The method of claim 1, wherein selecting said bacterium comprises use of a second binding polypeptide having specific affinity for the target ligand to label said target ligand bound to the candidate binding polypeptide.

4. The method of claim 3, wherein the second binding polypeptide is an antibody or fragment thereof.

5. The method of claim 4, wherein the antibody or fragment thereof is fluorescently labeled.

6. The method of claim 3, wherein selecting said bacterium comprises use of at least a third binding polypeptide having specific affinity for the target ligand and/or said second binding polypeptide to label said bacterium.

7. The method of claim 1, wherein the target ligand is fused to a detectable label.

8. The method of claim 7, wherein the detectable label is an antigen.

9. The method of claim 7, wherein the detectable label is GFP.

10. The method of claim 7, wherein the target ligand is further defined as fused to a cytoplasmic degradation signal.

11. The method of claim 10, wherein the cytoplasmic degradation signal is SsrA.

12. The method of claim 1, wherein said Gram negative bacterium is an E. Coli bacterium.

13. The method of claim 1, wherein step (a) is further defined as comprising providing a population of Gram negative bacteria.

14. The method of claim 13, wherein said population of bacteria is defined as collectively expressing nucleic acid sequences encoding a plurality of candidate binding polypeptides.

15. The method of claim 13, wherein said population of bacteria is further defined as collectively expressing nucleic acid sequences encoding a plurality of target ligands.

16. The method of claim 14, wherein the population of bacteria expresses a single target ligand.

17. The method of claim 13, wherein from about two to six rounds of selecting are carried out to obtain said bacterium from said population.

18. The method of claim 2, wherein the bacterium is non-viable.

19. The method of claim 2, wherein the bacterium is viable.

20. The method of claim 2, wherein cloning comprises amplification of the nucleic acid sequence.

21. The method of claim 1, wherein the candidate binding polypeptide is a fusion polypeptide.

22. The method of claim 1, wherein selecting is carried out by flow-cytometry or magnetic separation.

23. The method of claim 1, wherein said candidate binding polypeptide is further defined as an antibody or fragment thereof.

24. The method of claim 23, wherein said candidate binding polypeptide is further defined as a scAb, Fab or scFv.

25. The method of claim 1, wherein said candidate binding polypeptide is further defined as an enzyme.

26. The method of claim 1, wherein said target ligand is selected from the group consisting of a peptide, a polypeptide, an enzyme, a nucleic acid and a small molecule.

27. The method of claim 1, wherein said nucleic acid encoding a candidate binding polypeptide is flanked by known PCR primer sites.

28. The method of claim 1, wherein step (c) comprises permeabilizing and/or removing said outer membrane.

29. The method of claim 28, wherein permeabilizing and/or removing the outer membrane comprises a method selected from the group consisting of:

treatment with hyperosmotic conditions, treatment with physical stress, infecting the bacterium with a phage, treatment with lysozyme, treatment with EDTA, treatment with a digestive enzyme and treatment with a chemical that disrupts the outer membrane.

30. The method of claim 28, comprising removing the outer membrane.

31. The method of claim 29, wherein permeabilizing and/or removing the outer membrane comprises a combination of said methods.

32. The method of claim 31, wherein permeabilizing and/or removing the outer membrane comprises treatment with lysozyme and EDTA.

33. The method of claim 28, wherein permeabilizing and/or removing the outer membrane comprises treating the bacterium with a combination of physical, chemical and enzyme disruption of the outer membrane.

34. The method of claim 28, wherein said bacterium comprises a mutation conferring increased permeability of said outer membrane.

35. The method of claim 1, wherein step (c) comprises permeabilizing the outer membrane and washing the cell.

36. The method of claim 1, wherein said bacterium is grown at a sub-physiological temperature.

37. The method of claim 36, wherein said sub-physiological temperature is about 25°

C.

38. The method of claim 1, wherein said target ligand and said candidate binding polypeptide are reversibly bound.

39. The method of claim 1, wherein the target ligand is operably linked to a leader sequence capable of directing the export of the target ligand to the periplasm.

40. The method of claim 39, wherein the leader peptide is an ssTorA leader peptide.

41. The method of claim 1, wherein said inner membrane anchor polypeptide comprises a transmembrane protein or fragment thereof.

42. The method of claim 41, wherein the transmembrane protein or fragment thereof comprises a sequence selected from the group consisting of:

the first two amino acids encoded by the E. coli NlpA gene, the first six amino acids encoded by the E. coli NlpA gene, the gene III protein of filamentous phage or a fragment thereof, an inner membrane lipoprotein or fragment thereof.

43. The method of claim 41, wherein the inner membrane anchor polypeptide is fused to the candidate binding polypeptide via an N- or C-terminus.

44. The method of claim 1, wherein the inner membrane anchor polypeptide comprises an inner membrane lipoprotein or fragment thereof selected from the group consisting of:

AraH, MglC, MalF, MalG, Mal C, MalD, RbsC, RbsC, ArtM, ArtQ, GlnP, ProW, HisM, H is Q, LivH, LivM, LivA, Liv E,Dpp B, DppC, OppB,AmiC, AmiD, BtuC, FhuB, FecC, FecD,FecR, FepD, NikB, NikC, CysT, CysW, UgpA, UgpE, PstA, PstC, PotB, PotC,PotH, PotI, ModB, NosY, PhnM, LacY, SecY, TolC, DsbB, DsbD, TonB, TatC, CheY, TraB, Exb D, ExbB and Aas.

54. The method of claim 7, wherein the detectable label comprises the peptide sequence of SEQ ID NO:

33.

45. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:

(a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm;

said bacterium comprising a nucleic acid sequence encoding a candidate binding polypeptide, wherein the candidate binding polypeptide is anchored to the outer side of the inner membrane with an inner membrane anchor polypeptide;

wherein the bacterium further comprises a nucleic acid sequence encoding a target ligand, wherein the target ligand is exported to the periplasm;

(b) allowing the target ligand to bind to the candidate binding polypeptide;

(c) removing the outer membrane of said bacterium; and

(c) selecting the bacterium based on the presence of the target ligand bound to the candidate binding polypeptide on the outer side of the inner membrane.

46. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:

(a) providing a population of Gram negative bacteria the members of which comprise an inner membrane, an outer membrane and a periplasm;

said population collectively comprising nucleic acid sequences encoding plurality of candidate binding polypeptides, wherein the candidate binding polypeptides are anchored to the outer side of the inner membrane of said bacteria;

wherein the bacteria further comprise nucleic acid sequences encoding a target ligand, wherein the target ligand is exported to the periplasm;

(b) allowing the target ligand to bind to the candidate binding protein in said periplasm;

(c) removing the outer membrane of said bacterium; and

(d) selecting the bacterium from said population based on the presence of the target ligand bound to the candidate binding polypeptide on the outer side of the inner membrane.

47. The method of claim 46, wherein step (d) is further defined as selecting a subpopulation of bacteria comprising the target ligand bound to the candidate binding polypeptide.

48. The method of claim 46, wherein step (d) comprises fluorescently labeling said target ligand followed by fluorescence activated cell sorting (FACS).

49. A method of obtaining a bacterium comprising a nucleic acid sequence encoding a binding polypeptide having specific affinity for a target ligand comprising the steps of:

(a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm;

said bacterium comprising a nucleic acid sequence encoding a candidate binding polypeptide, wherein the candidate binding polypeptide is anchored to the outer side of the inner membrane;

wherein the bacterium further comprises a nucleic acid sequence encoding a fusion polypeptide comprising a target ligand, a periplasmic export signal, a fluorescent label and a cytoplasmic degradation signal;

(b) allowing the target ligand to bind to the candidate binding polypeptide;

(c) removing the outer membrane of said bacterium; and

(d) selecting the bacterium based on the presence of the target ligand bound to the candidate binding polypeptide on the outer side of the inner membrane using fluorescence activated cell sorting (FACS).

50. The method of claim 49, wherein the periplasmic export signal is TorA.

51. The method of claim 49, wherein the cytoplasmic degradation signal is SsrA.

52. The method of claim 49, wherein the fluorescent label is GFP.

53. The method of claim 49, wherein the fusion polypeptide comprises the following components from the N-terminus to C-terminus:

a periplasmic export signal, a target ligand, a fluorescent label and a cytoplasmic degradation signal.