Expression and characterization of a mutant recombinant blood coagulation factor VIII (rFVIII (m)).
In an earlier study, a site directed mutant rFVIII (rFVIII(m), Arg(336) --> Gln(336)) expressed in baculovirus-insect cell (Sf9) system was found to sustain high level activity during incubation at 37 degrees celsius for 24 h while the cofactor activity of normal plasma was declined steadily. In this study, a mutant B-domain deleted rFVIII(m), Arg(336) --> Gln(336) expressed in baculovirus-insect cell (Sf9) system was characterized for its enzymatic and chemical properties. The expressed rFVIII(m) and plasma FVIII (pFVIII) were purified by immunoaffinity column chromatography and identified by Western blot analysis. The partially purified rFVIII(m) exhibited cofactor specific activity of 2.01 x 10(3)units/mg protein. The molecular weight of rFVIII(m) ranged between 40 to 150 kDa with a major band at 150 kDa. Treatment of both rFVIII(m) and pFVIII with thrombin increased their cofactor activity in a similar pattern. Treatment of both the activated rFVIII(m) and native FVIII with APC decreased their cofactor activities, however, the former exhibited a slower decrease than the latter, although no significant difference was present. rFVIII(m) formed a complex with vWF, resulting in a stabilized form, and the lag period of thrombin-mediated activating was extended by vWF association. These results implicated that rFVIII(m) expressed in baculovirus-insect cell system had a comparable capacity as FVIII cofactor activity and might be a good candidate for the FVIII replacement therapy for hemophilia A patients.
Oh
Sang Hwan
SH
Department of Biochemistry and Molecular Biology, The Institue of Genetic Science, College of Medicine, Yonsei University, Seoul 120-752, Korea. shoh@yumc.yonsei.ac.kr
Cho
Sun Young
SY
Jin
Hyun Sun
HS
Jeong
Deuk-Lim
DL
Jung
Sung-Gil
SG
Oh
Keun Hee
KH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007313:Insecta; D046911:Macromolecular Substances; D009154:Mutation / Q000235:genetics*; D011486:Protein C / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism
D046911:Macromolecular Substances; D011486:Protein C; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C078147:F8 protein, human; D005169:Factor VIII; D013917:Thrombin