Methods for isolating a cDNA encoding a membrane-bound protein

• US 6,610,485 B1
• Filed: 05/02/2001
• Issued: 08/26/2003
• 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: Tsuchiya, Masayuki, Ohtomo, Toshihiko, Saito, Mikiyoshi
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/700,820
• Time in Patent Office: 846 Days
• Field of Search: 435/6, 435/810, 435/69.7
• US Class Current: 435/6.11
• 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 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 and kit disclosed herein are particularly useful for identifying genes for membrane-bound proteins represented in cDNA libraries.

Claims

1. A method for isolating or identifying a gene encoding a membrane-bound protein, the method comprising the steps of

(i) introducing into cells a vector comprising a sequence comprising (a) a DNA encoding a secretable protein and (b) a cDNA ligated downstream of the secretable protein-encoding DNA, wherein the secretable protein can bind to an antigen and is selected from the group consisting of an antibody, a fragment of an antibody, and a single-chain antibody;

(ii) expressing in the cells a fusion protein encoded by the sequence comprising (a) and (b);

(iii) contacting the cells of (ii) with the antigen;

(iv) selecting a cell that binds to the antigen; and

(v) isolating or identifying the cDNA comprised in the vector that was introduced into the selected cell.

2. The method of claim 1, further comprising determining the sequence of the cDNA isolated or identified in (v).

3. The method of claim 1, further comprising screening a cDNA library to obtain a full-length cDNA comprising the sequence of the cDNA isolated or identified in (v).

4. The method of claim 1, wherein the vector introduced into cells in step (i) is obtained by introducing a cDNA into a vector at a restriction enzyme site downstream of the 3′

end of the secretable protein-encoding DNA.

5. The method of claim 1, wherein the vector introduced into cells in step (i) is obtained by introducing into a vector, a DNA comprising (a) a DNA encoding the secretable protein and (b) a cDNA ligated downstream of the 3′

end of the secretable protein-encoding DNA.

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

7. The method of claim 1, wherein the cDNA is derived from a cDNA library obtained from mammalian cells.

8. The method of claim 1, wherein the vector introduced into cells in step (i) comprises a DNA encoding a secretion signal sequence upstream of the 5′

end of the DNA of (a).

9. The method of claim 1, wherein the secretable protein is an antibody.

10. The method of claim 1, wherein the secretable protein is a single-chain antibody.

11. The method of claim 10, wherein the vector contains a DNA encoding a constant region of an antibody ligated downstream of the 3′

end of the DNA encoding the single-chain antibody.

12. The method of claim 1, wherein the antigen is bound to a support.

13. The method of claim 12, wherein the support is for cell-culturing.