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Publication Detail
Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B
  • Publication Type:
    Journal article
  • Publication Sub Type:
  • Authors:
    Williams SC, Hinshelwood J, Perkins SJ, Sim RB
  • Publication date:
  • Pagination:
    625, 632
  • Journal:
    Biochemical Journal
  • Volume:
  • Print ISSN:
  • Keywords:
    recombinant, Complement Factor B, von Willebrand Factor, peptides, Peptide Fragments, Recombinant Proteins, Proteins, Acids, amino acid, Amino Acid Sequence, Amino Acids, Base Sequence, Binding Sites, Biochemistry, biosynthesis, cDNA, Complement 3b, Electrophoresis, Polyacrylamide Gel, Escherichia coli, genetics, isolation & purification, metabolism, Molecular Sequence Data, Mutation, Protein Binding, Serine, Solubility, Support, Non-U.S.Gov't, Thrombin, expression, Structure, function
  • Notes:
    UI - 99406315 LA - Eng RN - EC (Complement Factor B) RN - 0 (von Willebrand Factor) RN - 0 (Peptide Fragments) RN - 0 (Recombinant Proteins) RN - 80295-43-8 (Complement 3b) PT -JOURNAL ARTICLE DA - 19991208 IS - 0264-6021 SB - M SB - X CY -ENGLAND JC - 9YO AA - Author EM - 200002
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 C3b and properdin, and is activated by the protease factor D. The fourth domain 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 region 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 Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys(267)-->Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of (125)I-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 property of the vWF-A domain. The site at which factor D cleaves factor B is close 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 presence of C3b
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