Polypeptide display libraries and methods of making and using thereof

• US 7,612,019 B2
• Filed: 12/19/2006
• Issued: 11/03/2009
• 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.
• Primary Examiner(s): Steele; Amber D.
• Application Number:
  US11/612,757
• Publication Number:
  US 20070099247A1
• Time in Patent Office: 1,050 Days
• Field of Search: 506/2, 506/9, 506/11, 506/14, 435/243
• US Class Current: 506/9
• 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 assay method for detecting a target in a sample, said method comprising:

contacting a passenger polypeptide with a sample suspected of containing said target, wherein said passenger polypeptide is displayed on the surface of a biological entity, and wherein said passenger polypeptide is fused to an extracellular terminus of a circularly permuted bacterial outer membrane protein; and

detecting a specific interaction between the passenger polypeptide and the target when present, wherein presence of the specific interaction is indicative of the presence of the target in the sample.

2. The assay of claim 1, wherein the bacterial outer membrane protein is OmpA or OmpX.

3. The assay of claim 1, wherein the passenger polypeptide is a substrate for an enzyme.

4. The assay of claim 3, wherein the target is the enzyme.

5. The assay of claim 1, wherein the passenger polypeptide has a label.

6. The assay of claim 5, wherein the label is a fluorescent agent.

7. The assay of claim 5, wherein the passenger polypeptide has a C-terminal label.

8. The assay of claim 5, wherein the passenger polypeptide has an N-terminal label.

9. The assay of claim 5, wherein loss of the label indicates interaction between the passenger polypeptide and the target.

10. The assay of claim 1, wherein the assay further comprises quantifying the biological entity by flow cytometry, fluorescence, or microscopy.

11. The assay of claim 1, wherein the biological entity expresses a fluorescent molecule or a luminescent reporter molecule.

12. The assay of claim 1, wherein the target is associated with a second biological entity or a particle.

13. The assay of claim 12, wherein interaction between the passenger polypeptide and the target results in a complex between the biological entity and the second biological entity or the particle.

14. The assay of claim 13, wherein the complex is quantified by flow cytometry, fluorescence, or microscopy.

15. The assay of claim 1, wherein the passenger polypeptide comprises SEQ ID NO:

315.

16. The assay of claim 15, wherein the passenger polypeptide is capable of binding C-reactive protein.

17. The assay of claim 1, wherein the passenger polypeptide comprises SEQ ID NO:

99, SEQ ID NO;

210, SEQ ID NO;

211, SEQ ID NO;

212, SEQ ID NO;

213, SEQ TD NO;

214, SEQ ID NO;

215, SEQ ID NO;

216, SEQ ID NO;

217, SEQ ID NO;

218, SEQ ID NO;

219, SEQ ID NO;

220, SEQ ID NO;

221, SEQ ID NO;

222, SEQ ID NO;

223, SEQ ID NO;

224, SEQ ID NO;

225, SEQ ID NO;

226, SEQ ID NO;

227, SEQ ID NO;

228, SEQ ID NO;

229, SEQ ID NO;

230, SEQ ID NO;

231, SEQ ID NO;

232, SEQ ID NO;

233, SEQ ID NO;

234, SEQ ID NO;

235, SEQ ID NO;

236, SEQ ID NO;

237, SEQ ID NO;

238, SEQ ID NO;

239, SEQ ID NO;

240, SEQ ID NO;

241, SEQ ID NO;

242, SEQ ID NO;

243, SEQ ID NO;

244, SEQ ID NO;

245, SEQ ID NO;

246, SEQ ID NO;

247, SEQ ID NO;

248, SEQ ID NO;

249, SEQ ID NO;

250, SEQ ID NO;

251, SEQ ID NO;

252, SEQ ID NO;

253, SEQ ID NO;

254, or SEQ ID NO;

256.

18. The assay of claim 1, wherein the passenger polypeptide consists of SEQ ID NO:

99, SEQ ID NO;

210, SEQ ID NO;

211, SEQ ID NO;

212, SEQ ID NO;

213, SEQ ID NO;

214, SEQ ID NO;

215, SEQ ID NO;

216, SEQ ID NO;

217, SEQ ID NO;

218, SEQ ID NO;

219, SEQ ID NO;

220, SEQ ID NO;

221, SEQ ID NO;

222, SEQ ID NO;

223, SEQ ID NO;

224, SEQ ID NO;

225, SEQ ID NO;

226, SEQ ID NO;

227, SEQ ID NO;

228, SEQ ID NO;

229, SEQ ID NO;

230, SEQ ID NO;

231, SEQ ID NO;

232, SEQ ID NO;

233, SEQ ID NO;

234, SEQ ID NO;

235, SEQ ID NO;

236, SEQ ID NO;

237, SEQ ID NO;

238, SEQ ID NO;

239, SEQ ID NO;

240, SEQ ID NO;

241, SEQ ID NO;

242, SEQ ID NO;

243, SEQ ID NO;

244, SEQ ID NO;

245, SEQ ID NO;

246, SEQ ID NO;

247, SEQ ID NO;

248, SEQ ID NO;

249, SEQ ID NO;

250, SEQ ID NO;

251, SEQ ID NO;

252, SEQ ID NO;

253, SEQ ID NO;

254, or SEQ ID NO;

256.