Single chain antibody (SCA) encoding genes: one-step construction and expression in eukaryotic cells.

Abstract

We report the expression, in eukaryotic cells, of a gene encoding a single chain antibody (SCA) and a rapid method for the construction of such genes. A SCA directed against the aromatic dye fluorescein was synthesized from a gene constructed by means of the simultaneous use of four PCR primers and templates of both light and heavy chain immunoglobulin cDNAs in the form of either plasmid clones or reverse transcribed hybridoma RNA. Two of the primers were partially complementary to one another and encoded the polypeptide linker which joins the immunoglobulin light and heavy chain variable domains of the SCA polypeptide. A functional, hapten-binding product was synthesized from the gene thus constructed in both E. coli and the fission yeast, Schizosaccharomyces pombe. Our results demonstrate that gene constructs encoding single chain antigen binding proteins can be synthesized very rapidly with only limited sequence information about the pertinent light and heavy chain immunoglobulin genes, and, that neither murine codon usage bias, Thermus aquaticus DNA polymerase infidelity, nor the eukaryotic cellular environment preclude the synthesis of functional single chain antigen binding proteins in non-lymphatic, non-murine eukaryotic cells.