Methods for producing members of specific binding pairs

Abstract

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

1 . A phagemid comprising DNA encoding a polypeptide-coliphage pIII fusion protein, wherein said fusion protein comprises a single-chain polypeptide and a functional coliphage pIII polypeptide, and said functional coliphage pIII polypeptide comprises contiguous amino and carboxy domains of a coliphage pIII protein. 
     
     
         2 . A phagemid according to  claim 1 , wherein the single-chain polypeptide is a single-chain antibody. 
     
     
         3 . A phagemid according to  claim 1 , wherein said polypeptide-coliphage pIII fusion protein contains a protease-sensitive site between the single-chain polypeptide and the coliphage pIII polypeptide. 
     
     
         4 . A phagemid according to  claim 1  or  claim 2 , further comprising expression control elements upstream of said DNA and further encoding at least one selectable marker. 
     
     
         5 . A process for the production of a phagemid according to  claim 1 , comprising fusing a DNA encoding a single-chain polypeptide to a DNA encoding a functional coliphage pIII polypeptide, wherein said functional coliphage pIII polypeptide comprises contiguous amino and carboxy domains of a coliphage pIII protein, and inserting the resulting DNA molecule into a phagemid. 
     
     
         6 . The process according to  claim 5 , further comprising inserting a protease-sensitive site between the DNA encoding the single-chain polypeptide and the DNA encoding the coliphage pIII polypeptide. 
     
     
         7 . A phagemid according to  claim 1 , wherein said coliphage pIII polypeptide is a full-length coliphage pIII protein. 
     
     
         8 . A method of screening for binding ligands, comprising exposing ligands to a single chain polypeptide-coliphage pIII protein expressed by the phagemid of  claim 1  and selecting those ligands which recognize and bind to the single chain polypeptide-coliphage pIII protein.