IN-VIVO BIOTINYLATED RECOMBINANT ANTIBODIES - CONSTRUCTION, CHARACTERIZATION, AND APPLICATION OF A BIFUNCTIONAL FAB-BCCP FUSION PROTEIN PRODUCED IN ESCHERICHIA-COLI

We describe a novel vector system suitable for the efficient preparati on of in vivo biotinylated antibody Fab fragments in Escherichia coli. The previously described pGE20 vector used for the functional express ion of truncated heavy (Fd) and light (L) chains of Fab into the bacte rial culture medium was modified by inserting the C-terminal 101-amino -acid polypeptide of the biotin carboxyl carrier protein subunit of E. coli acetyl-CoA carboxylase (BCCP). The secreted Fd-BCCP* fusion and L chain proteins were found to be disulfide linked and Fab-BCCP comp lexes of an IgG1 antibody (Mab4) to human tumor necrosis factor alpha (TNF) were shown to retain both antigen and streptavidin-binding activ ities. The capacity of the Fab4 linked to BCCP to bind TNF was identi cal to that observed with unmodified Fab4. Up to 15% of the expressed hybrids were able to interact with streptavidin when exogeneous d-biot in was added into the bacterial culture medium. The Fab4-BCCP molecul es were found to be more efficient than Fab4 linked to an engineered s treptavidin-affinity tag for the detection of antigen on solid phase. In addition, we show here that the bacterially expressed Fab4-BCCP co mplexes, adsorbed to streptavidin-agarose beads, can be used for the o ne-step purification of recombinant TNF by immunoaffinity chromatograp hy. (C) 1994 Academic Press, Inc.