Compositions for isolating a cDNA encoding a membrane-bound protein

• US 7,338,797 B2
• Filed: 04/29/2003
• Issued: 03/04/2008
• Est. Priority Date: 05/20/1998
• Status: Expired due to Fees

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: Saito, Mikiyoshi, Tsuchiya, Masayuki, Ohtomo, Toshihiko
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US10/425,529
• Publication Number:
  US 20030175794A1
• Time in Patent Office: 1,771 Days
• Field of Search: 435/320.1
• US Class Current: 435/320.1
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors

Abstract

A method, vector, and kit are provided for selectively isolating genes encoding membrane-bound proteins by fusing proteins to a secretable protein having a binding affinity to an antigen (e.g., an antibody), expressing these fusions in host cells, and selectively isolating individual host cells by their ability to bind the antigen via the fusion protein. Host cells expressing fusion proteins that do not contain a membrane binding domain will be relatively unable to bind the antigen, since the fusion proteins in those cells are secreted and unattached to their host cells. The method, vector, and kit disclosed herein are particularly useful for identifying genes for membrane-bound proteins represented in cDNA libraries.

Claims

1. A cDNA library comprising a mixture of vectors, each of which comprises (a) a DNA encoding a secretable protein that binds to an antigen and (b) a cDNA ligated at the 3′

end of the DNA encoding the secretable protein, wherein the secretable protein is invariable for all of the vectors in the library and is selected from the group consisting of an antibody, a fragment thereof, and a single-chain antibody, and wherein the cDNA in at least one of the vectors encodes a membrane-bound protein.

2. The cDNA library of claim 1, wherein the cDNA is inserted at a restriction enzyme site downstream of the 3′

end of the DNA encoding the secretable protein.

3. The cDNA library of claim 1, wherein the DNA of (a) and the cDNA of (b) are ligated via a DNA encoding a peptide linker.

4. The cDNA library of claim 1, wherein the cDNA is derived from mammalian cells.

5. The cDNA library of claim 1, wherein the secretable protein comprises a secretion signal.

6. The cDNA library of claim 1, wherein the secretable protein comprises a single-chain antibody.

7. The cDNA library of claim 6, wherein the secretable protein further comprises a constant region of an antibody located at the C-terminal end of the single-chain antibody.

8. The cDNA library of claim 6, wherein the single-chain antibody is bivalent.

9. A kit for isolating a cDNA encoding a membrane-bound protein, wherein the kit comprises:

(i) the cDNA library according to claim 1; and

(ii) the antigen.

10. The kit of claim 9, wherein the antigen is bound to a support.

11. The kit of claim 9, further comprising cells into which the vectors can be introduced.

12. The kit of claim 9, further comprising a cross-linking agent.

13. The cDNA library of claim 1, wherein the membrane-bound protein is selected from the group consisting of type I membrane-bound proteins, type II membrane-bound proteins, and GPI anchor-type membrane-bound proteins.

14. The cDNA library of claim 1, wherein the membrane-bound protein has multiple transmembrane regions.

15. The cDNA library of claim 1, wherein the membrane-bound protein is a mitochondrial inner-membrane protein.

16. The cDNA library of claim 1, wherein the membrane-bound protein is selected from the group consisting of cytochrome oxidase, NADH-dehydrogenase, and ATP-synthase.

17. The cDNA library of claim 1, wherein the antigen is selected from the group consisting of serum proteins, cytokines, and intracellular proteins.

18. The cDNA library of claim 1, wherein the antigen is an interleukin 6 receptor.

19. The cDNA library of claim 1, wherein the cDNA library is obtained by a process comprising the steps of:

(a) isolating a mixture of mRNAs from cells, embryos, tissues, blood, or organs;

(b) synthesizing a mixture of cDNAs from the mRNAs; and

(c) incorporating the cDNAs into vectors such that each vector comprises (i) a DNA encoding the secretable protein and (ii) a cDNA ligated at the 3′

end of the DNA encoding the secretable protein.

20. Cells transformed with the cDNA library of claim 1.