Polypeptide Display Libraries and Methods of Making and Using Thereof

• US 20070099247A1
• Filed: 12/19/2006
• Published: 05/03/2007
• 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, Bessette, Paul
• Granted Patent: US 7,612,019 B2
• Time in Patent Office: Days
• US Class Current: 435/7.2
• 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, monitoring, or measuring a given ligand in a sample which comprises

inducing an expression vector capable of genetically expressing and displaying a given passenger polypeptide in a carrier protein that is an extracellular loop of a bacterial membrane protein to result in (a) the passenger polypeptide being capable of interacting with the ligand, and (b) a terminus of the extracellular loop, on an outer surface of a biological entity to express the passenger polypeptide and then

contacting the passenger polypeptide with the sample and

observing whether the passenger polypeptide interacts with the ligand.

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

3. The assay of claim 2, wherein the ligand is the enzyme.

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

5. The assay of claim 4, wherein the label is a fluorescent agent.

6. The assay of claim 4, wherein the free terminus of the passenger polypeptide has the label.

7. The assay of claim 1, wherein loss of the label being detectable indicates interaction between the passenger polypeptide and the ligand.

8. The assay of claim 1, wherein the biological entity is quantified by flow cytometry, fluorescence, or microscopy.

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

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

11. The assay of claim 10, wherein interaction between the passenger polypeptide and the ligand results in a complex between the biological entity and the second biological entity or the particle.

12. The assay of claim 11, wherein the complex is quantified by flow cytometry, fluorescence, or microscopy.

13. The assay of claim 1, wherein the passenger polypeptide comprises NXRGF, wherein X is any amino acid.

14. The assay of claim 13, wherein the passenger polypeptide is capable of binding C-reactive protein.

15. 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 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.

16. 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.

17. An isolated polypeptide comprises NXRGF, wherein X is any amino acid.

18. The isolated polypeptide of claim 17, wherein the polypeptide is capable of binding C-reactive protein.

19. The isolated polypeptide of claim 17, 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 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.

20. The isolated polypeptide of claim 17, 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.