Compositions for isolating a cDNA encoding a membrane-bound protein

Abstract

A method for isolating a gene encoding a membrane-bound protein characterized by fusing a functional protein with a fused protein itself, which differs from the existing TMT method in which an epitope recognized by an antibody is carried in a fused protein. This method enabled selective isolation of a gene encoding a membrane-bound protein.

1 . (Canceled)  
     
     
         2 . The method of  claim 17 , 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.  
     
     
         3 . The method of  claim 17 , 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.  
     
     
         4 . The method of  claim 17 , wherein the DNA of (a) and the cDNA of (b) are ligated via a DNA encoding a peptide linker.  
     
     
         5 . The method of  claim 17 , wherein the cDNA is derived from a cDNA library obtained from mammalian cells.  
     
     
         6 . The method of  claim 17 , 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).  
     
     
         7 . The method of  claim 17 , wherein the secretable protein is an antibody.  
     
     
         8 . The method of  claim 17 , wherein the secretable protein is a single-chain antibody.  
     
     
         9 . The method of  claim 8 , 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.  
     
     
         10 . The method of  claim 17 , wherein the antigen is bound to a support.  
     
     
         11 . The method of  claim 10 , wherein the support is for cell-culturing.  
     
     
         12 .- 16 . (Canceled)  
     
     
         17 . 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.    
     
     
         18 . The method of  claim 17 , further comprising determining the sequence of the cDNA isolated or identified in (v).  
     
     
         19 . The method of  claim 17 , further comprising screening a cDNA library to obtain a full-length cDNA comprising the sequence of the cDNA isolated or identified in (v).