Method of identifying a gene product

• US 6,861,229 B2
• Filed: 01/03/2003
• Issued: 03/01/2005
• Est. Priority Date: 07/06/2000
• Status: Active Grant

• Current Assignee: GenVec, Inc. (Precigen, Inc.)
• Original Assignee: GenVec, Inc. (Precigen, Inc.)
• Inventors: Carrión, Miguel E., Kovesdi, Imre
• Primary Examiner(s): McKelvey, Terry
• Assistant Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US10/336,702
• Publication Number:
  US 20030143609A1
• Time in Patent Office: 788 Days
• Field of Search: 435/7.1, 435/455, 435/456, 435/471, 435/6, 435/320.1
• US Class Current: 435/7.1
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2563/167 Mass label

Abstract

The present invention provides a method of identifying a gene product. The method comprises providing a multiplicity of cells comprising a first gene product. Preferably, the first gene product is produced in the multiplicity of cells by expressing a first exogenous nucleic acid sequence encoding the first gene product. A library of second nucleic acid sequences encoding second gene products is then introduced into the multiplicity of cells. The second nucleic acid sequences are expressed in the multiplicity of cells to produce the second gene products such that the first gene product and at least one of the second gene products contact. The method further comprises causing a complex to form between the first gene product, an affinity molecule that binds the first gene product, and at least one of the second gene products, and subsequently retrieving the complex. At least one second gene product of the complex then is identified.

Claims

1. A method of identifying a gene product, wherein the method comprises:

(a) providing a multiplicity of cells comprising a first gene product;

(b) introducing into the multiplicity of cells a library of second nucleic acid sequences encoding second gene products;

(c) expressing the second nucleic acid sequences to produce the second gene products such that the first gene product and at least one of the second gene products are secreted from the multiplicity of cells, and the first gene product and at least one of the second gene products contact extracellularly;

(d) causing at least one complex to form between the first gene product, an affinity molecule, and at least one of the second gene products;

(e) retrieving the complex; and

(f) identifying at least one second gene product of the complex, wherein identifying at least one second gene product of the complex comprises;

(f1) producing charged fragments from at least a portion of the complex;

(f2) detecting the charged fragments by a detector which produces a signal corresponding to the mass-to-charge ratio of the charged fragments and comprises information about the amino acid sequence of at least one second gene product; and

(f3) evaluating the signal to identify the at least one of the second gene products.

2. The method of claim 1, wherein the first gene product is produced in the multiplicity of cells via expression of a first exogenous nucleic acid sequence encoding the first gene product.

3. The method of claim 2, wherein the first nucleic acid sequence is present in a plasmid.

4. The method of claim 2, wherein the first nucleic acid sequence is present in a viral vector.

5. The method of claim 4, wherein the viral vector is an adenoviral vector.

6. The method of claim 2, wherein the second nucleic acid sequence is present in a plasmid.

7. The method of claim 2, wherein the second nucleic acid sequence is present in a viral vector.

8. The method of claim 7, wherein the viral vector is an adenoviral vector.

9. The method of claim 1, wherein identifying at least one second gene product of the complex comprises dissociating the complex between the first gene product, the affinity molecule, and at least one of the second gene products such that the first gene product and at least one of the second gene products remain intact.

10. The method of claim 1, wherein the affinity molecule is fixed to a solid support.

11. The method of claim 10, wherein the solid support is a bead or an affinity column.

12. The method of claim 10, wherein identifying at least one second gene product of the complex comprises dissociating the complex between the first gene product, the affinity molecule, and at least one of the second gene products such that the first gene product and at least one of the second gene products remain intact.

13. The method of claim 1, further comprising (g) identifying the second nucleic acid sequence encoding at least one of the second gene products.

14. The method of claim 1, wherein the first gene product comprises a heterologous portion which associates with an affinity molecule.

15. A method of identifying a gene product, wherein the method comprises:

(a) providing a viral vector comprising a first nucleic acid sequence encoding a first gene product;

(b) providing a library of viral vectors, wherein each viral vector of the library comprises a second nucleic acid sequence encoding a second gene product;

(c) transducing a multiplicity of host cells with the viral vector comprising the first nucleic acid sequence and the library of viral vectors comprising second nucleic acid sequences, wherein the host cells are permissive for expression of the first nucleic acid sequence and the second nucleic acid sequences and production of the first gene product and the second gene products;

(d) expressing the first nucleic acid sequence and the second nucleic acid sequences such that the first gene product and at least one of the second gene products are secreted from the multiplicity of cells, and the first gene product and at least one of the second gene products contact extracellularly;

(e) causing at least one complex to form between the first gene product, an affinity molecule, and at least one of the second gene products;

(f) retrieving the formed complex; and

(g) identifying at least one second gene product of the complex.

16. The method of claim 15, wherein the first gene product comprises a heterologous portion which associates with an affinity molecule.

17. The method of claim 15, wherein the viral vectors are adenoviral vectors.

18. The method of claim 15, wherein identifying at least one second gene product of the complex comprises:

(g1) producing charged fragments from at least a portion of the complex;

(g2) detecting the charged fragments by a detector which produces a signal corresponding to the mass-to-charge ratio of the charged fragments and comprising information about the amino acid sequence of at least one of the second gene products; and

(g3) evaluating the signal to identify at least one of the second gene products.