Expression of biologically active human factor VIII using a baculovirus vector.
Factor VIII is a complex, plasma glycoprotein involved in the process of blood coagulation. Production of the recombinant molecule has largely been confined to mammalian cell systems which have, in general, proven to be inefficient producers of factor VIII. The use of a baculovirus expression system may provide increased levels of this glycoprotein, although it is not certain that insect cell-derived factor VIII will be biologically active. The N-linked glycosylation patterns in insect cells, until recently thought to be less complex than in mammalian cells, may influence activity and/or secretory ability. To this end we engineered a B domain-deleted factor VIII cDNA sequence for expression in Spodoptera frugiperda cells. The construct retained the native signal sequence to allow secretion of recombinant protein into the culture medium. Initial studies revealed the production of secreted factor VIII, and this protein was shown to possess coagulation activity. The presence of N-linked oligosaccharide residues was demonstrated, the glycosylated molecule being of a similar size to that expressed in mammalian cells.
Webb
E
E
Research and Development Division, CSL Limited, Parkville, Victoria, Australia.
Tkalcevic
J
J
Edwards
S
S
Hocking
D
D
Nisbet
I
I
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001777:Blood Coagulation; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
D011994:Recombinant Proteins; D005169:Factor VIII; D004247:DNA