APPLICATION OF AN ALKALINE-PHOSPHATASE FUSION PROTEIN SYSTEM SUITABLEFOR EFFICIENT SCREENING AND PRODUCTION OF FAB-ENZYME CONJUGATES IN ESCHERICHIA-COLI

We report a novel vector system suitable for the efficient preparation of alkaline phosphatase (PhoA)-labelled antibody Fab fragments in Esc herichia coli. The previously described pGE20 vector used for the func tional expression of truncated heavy (Fd) and light (L) chains of Fab into the bacterial culture medium was modified by inserting the E. col i PhoA coding region 3' to the Fd cloning sites. The secreted Fd-PhoA fusions and L proteins were found to be disulfide linked and Fab-PhoA complexes, prepared with IgG1 antibodies recognizing specifically huma n tumor necrosis factor a, were shown to be useful for the rapid detec tion of antigen. When an additional short peptide was interposed betwe en the Fd and PhoA domains, nearly all of the recombinant Fab-PhoA con jugates present in the culture supernatant retained both antigen bindi ng and enzymatic activity. A method for the detection and selection of bacterial colonies expressing bifunctional hybrid molecules of desire d antigen specificity was also developed. Taken together, the systems described permit the generation and production of Fab-PhoA conjugates in E. coli, which can replace conventionally prepared PhoA-labelled an tibodies in appropriate immunoassays.