Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B

Factor B is a five-domain 90 kDa serine protease proenzyme which:is part of the human serum complement system. It binds to other complement proteins C 3b and properdin, and is activated by the protease factor D. The fourth dom ain of factor B is homologous to the type A domain of von Willebrand Factor (VWF-A). A full-length human factor B cDNA clone was used to amplify the r egion encoding the VWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Es cherichia coli, where thrombin cleavage was used to separate the vWF-A doma in from its fusion protein partner. A second VWF-A domain with improved sta bility and solubility was created using a Cys(267) --> Ser mutation and a f our-residue C-terminal extension of the first VWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density ce ntrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consiste nt with known VWF-A crystal structures. This expression system and its char acterization permitted the first investigation of the function of the isola ted VWF-A domain. It was able to inhibit substantially the binding of I-125 -labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding prop erty of the VWF-A domain. The site at which factor D cleaves factor B is cl ose to the N-terminus of both recombinant vWF-A:domains. Factor D was shown to cleave the VWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the pr esence of C3b.