Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule

Abstract

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.

Mapping of the C1q Binding Site on Rituxan, a Chimeric Antibody with a Human IgG1 Fc

“…The chimeric L and H chains of Rituximab (IDEC Pharmaceuticals, San Diego, CA) (1) subcloned separately into previously described pRK vectors (19) were used. By site-directed mutagenesis (20), alanine variants of the C H 2 domain of the H chain were constructed.…”

“…Consortium, Huntsville, AL). Each polypeptide was subcloned separately into previously described pRK vectors (19) and cotransfected into human embryonic kidney 293 cells. The human FcRn was expressed as a His 6 -tagged extracellular domain, purified by Ni-NTA column (Qiagen, Valencia, CA) chromatography, and buffer exchanged into PBS.…”

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

“…Cleavage after residue Arg-740 releases the heavily glycosylated B domain. Previous studies demonstrated that the B domain of FVIII is dispensable for FVIII cofactor activity (22)(23)(24). Genetically engineered FVIII molecules that have varying degrees of B domain deletion yield functional FVIII molecules that are more efficiently expressed in mammalian cells (22,23,25,26).…”

“…Previous studies demonstrated that the B domain of FVIII is dispensable for FVIII cofactor activity (22)(23)(24). Genetically engineered FVIII molecules that have varying degrees of B domain deletion yield functional FVIII molecules that are more efficiently expressed in mammalian cells (22,23,25,26). These deletion molecules exhibit typical thrombin activation that correlates with cleavage after Arg-372, Arg-740, and Arg-1689, generating a FVIIIa heterotrimer that is indistinguishable from wild-type FVIII (FVIII WT) and also is subject to rapid inactivation though dissociation of the A2 domain subunit.…”

Protein engineering. The design, synthesis and characterization of factitious proteins

“…Yeast phosphoglycerate kinase has been used with some success to show that the model-of a substrateinduced change from the 'open' to the 'closed' form of the enzyme may need revision and will certainly profit from further study by directed mutagenesis (Wilson et al, 1987). Phosphoglycerate kinase has also been subjected to domain 'shuffling', a topic which is certain to become of consuming interest for very large multi-domain polypeptides such as Factor VIII where as much as one-third of the protein may be removed without important consequences for function (Eaton et al, 1986). The phosphoglycerate kinase experiment involved the successful construction of a chimeric protein with one domain from the human enzyme and the other from yeast (Mas et al, 1986).…”