Secretion of biologically active recombinant fibrinogen by yeast

Abstract

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (Aα, 67 kDa; Bβ, 57 kDa; γ, 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor Fα1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Mats his3-Δ1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 x 108 cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 μg/ml of fibrinogen into the culture medium. The Bβ and y chains, but not Aα, were glycosylated. Glycosylation of Bβ and γ, chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular Bβ and γ, chains, but not the Aα chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to he biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo γ chain dimerization and Aα chain polymer formation.