The interaction of the calcium- and integrin-binding protein (CIBP) with the coagulation factor VIII.
The gene encoding the C-terminal part of A1-domain of human blood coagulation factor VIII (FVIII), a 110-amino acid fragment from Ala(227) to Arg(336), namely A1(Delta1-226), was cloned and used as a 'bait' to screen a protein, which might interact with this region by using the yeast two-hybrid system. A gene coding for a related protein of FVIII named calcium- and integrin-binding protein (CIBP) was isolated from the normal human liver cDNA library. The results were confirmed by using the mammalian two-hybrid system and coimmunoprecipitation. The gene coding for CIBP was constructed by polymerase chain reaction (PCR) and then cotransfected with the B-domain-deleted FVIII gene into mammalian cell lines using the expression vector of FVIII for transient or stable expression. The culture supernatant was collected and analyzed both by enzyme-linked immunosorbent assay (ELISA) for FVIII antigen level and by one-stage method for procoagulant activity. Coexpressed with CIBP, the antigen level of FVIII in the mammalian cell line baby hamster kidney (BHK) cells increased up to about 170% and its bioactivity rose accordingly.
Fang
X
X
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, 320 Yue Yang Road, 200031, Shanghai, People's Republic of China.
Chen
C
C
Wang
Q
Q
Gu
J
J
Chi
C
C
D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D008099:Liver; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques
C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; D005169:Factor VIII