Expression and characterization of a mutant recombinant blood coagulation factor VIII (rFVIII (m))
Experimental & Molecular Medicine
;
: 233-238, 2002.
Artigo
em Inglês
| WPRIM
| ID: wpr-198789
ABSTRACT
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 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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Proteínas Recombinantes
/
Fator VIII
/
Fator de von Willebrand
/
Proteína C
/
Trombina
/
Linhagem Celular
/
Baculoviridae
/
Substâncias Macromoleculares
/
Insetos
/
Mutação
Tipo de estudo:
Estudo prognóstico
Limite:
Animais
Idioma:
Inglês
Revista:
Experimental & Molecular Medicine
Ano de publicação:
2002
Tipo de documento:
Artigo
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