SECRETION OF BIOLOGICALLY-ACTIVE RECOMBINANT FIBRINOGEN BY YEAST

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting, Human fibrinogen is a dimer wit h each half molecule com posed of three different polypeptides (A alph a, 67 kDa; B beta, 57 kDa; gamma, 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system cap able of producing substantial amounts of fibrinogen for structure func tion studies, we developed a recombinant system capable of secreting f ibrinogen. An expression vector (pYES2) was constructed with individua l fibrinogen chain cDNAs under the control of a Gal-1 promoter fused w ith mating factor F alpha 1 prepro secretion signal (SS) cascade. In a ddition, other constructs were prepared with combinations of cDNAs enc oding two chains or all three chains in tandem. Each chain was under t he control of the Gal-1 promoter. These constructs were used to transf orm Saccharomyces cerevisiae (INVSC1; Mat alpha his3-Delta 1 leu2 trp1 -289 ura3-52) in selective media. Single colonies from transformed yea st cells were grown in synthetic media with 4% raffinose to a density of 1 x 10(8) cells/ml and induced with 2% galactose for 16 h. Yeast ce lls expressing all three chains contained fibrinogen precursors and na scent fibrinogen and secreted about 30 mu g/ml of fibrinogen into the culture medium. The B beta and gamma chains, but not A alpha, were gly cosylated. Glycosylation of B beta and gamma chains was inhibited by t reatment of transformed yeast cells with tunicamycin. Intracellular B beta and gamma chains, but not the A alpha chains in secreted fibrinog en, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galact osylated biantennary oligosaccharide, Recombinant fibrinogen yielded t he characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrin ogen was shown to be biologi cally active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo gamma chain dimerization a nd A alpha chain polymer formation.