Peptide affinity chromatography of human clotting factor VIII. Screening of the vWF-binding domain.
The region of von Willebrand factor, which is involved in the complex formation with factor VIII, was used to generate a panel of octapeptides. A peptide ladder was generated from the von Willebrand factor region aa40 to aa100 and was synthesized on cellulose membranes by spot technology. Four peptides with affinity for factor VIII were identified by incubation with plasma derived factor VIII and recombinant factor VIII. The peptides denoted as 010 (LCPPGMVRHE), 011 (RCPCFHQGK), 014 (CFHQGKEYA) and 015 (RDRKWNCTDHVC) were further characterized by real-time interaction analysis and small scale affinity chromatography. Biotinylated peptides were used for blotting assays. These experiments showed that the peptides are directed against the light chain of FVIII. We consider these peptides as valuable tools for in situ labeling and also as ligands suitable for affinity chromatography.
Necina
R
R
Institute of Applied Microbiology, University of Agriculture, Forestry and Biotechnology, Vienna, Austria.
Amatschek
K
K
Schallaun
E
E
Schwinn
H
H
Josic
D
D
Jungbauer
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D014841:von Willebrand Factor / Q000378:metabolism*
D014841:von Willebrand Factor; D005169:Factor VIII