PRODUCTION AND SECRETION OF A BIFUNCTIONAL STAPHYLOCOCCAL PROTEIN-A-ANTIPHYTOCHROME SINGLE-CHAIN FV FUSION PROTEIN IN ESCHERICHIA-COLI

A bifunctional molecule was genetically engineered which contained the secretory signal and four Fc-binding domains of Staphylococcus aureus protein A (FcA), fused to a single-chain Fv (scFv) derived from an immunoglobulin (1g) G1 mouse monoclonal antibody (AS32) directed against the plant regulatory photoreceptor protein, phytochrome. The FcA=AS32scFv sequence was encoded in a single synthetic gene and expressed as a 60-kDa periplasmic protein in Escherichia coli. The bifunctionality of the fusion protein was established by its ability to bind to both IgG-agarose and phytochrome-sepharose. Growth of cultures, producing the FcA=AS32scFv, at 37-degrees-C, resulted in a decrease in the periplasmic accumulation of the fusion protein, and an increased accumulation of an assumed degradation product which retained Fc-binding activity. Growth of cultures at lower temperatures favoured the accumulation of undegraded fusion protein. The recombinant fusion protein could be purified to homogeneity by a simple, rapid chromatography procedure.