Novel forms of B-domain-deleted recombinant factor VIII molecules Construction and biochemical characterization

摘要: Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and 90-kDa polypeptide chain lacking B domain, have been produced using various cDNA constructs in order obtain primary translation products which were efficiently processed into 80 + complex. Three types single-chain cDNAs encoding B-domain-deleted derivatives designed, taking account sites at Arg740 Glu1649, assumed be important for processing VIII. In type 1 constructs, either Arg747, Arg752, or Arg776 N-terminal region domain was fused N-terminus (Glu1649) subunit. 2 construct r-VIII SQ, Ser743 Gln1638, creating link 14 amino acids between C-terminus (Arg740) chain, whereas RH, Arg747 His1646. 3 B-domain completely removed replaced with 1-4 Arg residues. After expression Chinese hamster ovary cells, 0-2 residues inserted found only partially contained large amount 170-kDa product. contrast, most SQ RH R4 R5 containing three four extra preceding desired complexes. The feature common deletion that they recognition motif proteolytic cleavage by membrane-bound subtilisin-like protease furin, is expressed cells; is, basic acid positions -1 -4 relative site Glu1649. Biochemical studies R5, two effectively derivatives, showed both proteins had normal cofactor function, N- C-termini chains corresponding those