Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale.
Recombinant von Willebrand factor (r-vWF) was produced in serum-free medium on a large scale in recombinant Chinese hamster ovary cells and was purified from fermentation supernatant by a combination of anion exchange chromatography and heparin affinity chromatography. Heparin affinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative functional analysis. We could show that while binding of r-vWF to platelets did not depend on multimerization of the molecule, ristocetin-induced platelet aggregation, binding to collagen and binding to heparin correlated directly with the extent of multimerization. Binding of recombinant coagulation factor VIII (r-FVIII) to r-vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The data indicated that binding of r-FVIII did not depend on r-vWF multimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecule. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding rate constant of 3 x 10(6) M-1 s-1 and an association constant of 2.5 x 10(9) M-1. Reaction of r-vWF with carbohydrate-specific lectins demonstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal sialic acid. Carbohydrate moities were covalently bound to the protein structure and were quantitatively removed from r-vWF only after protein denaturation. The results demonstrated that r-vWF produced on large scale under serum-free culture conditions exhibited qualitative and quantitative functional properties comparable to human plasma-derived vWF.
Fischer
B E
BE
IMMUNO AG, Biomedical Research Center, Orth an der Donau, Austria.
Schlokat
U
U
Reiter
M
M
Mundt
W
W
Dorner
F
F
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D006001:Glycoconjugates / Q000032:analysis; D006801:Humans; D007158:Immunologic Techniques; D046911:Macromolecular Substances; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
D006001:Glycoconjugates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin