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Biomedical subjects
I Nisbet
Publications and source records attributed to I Nisbet.
Enhanced secretory ability for the human factor VIII light chain produced in baculovirus-infected insect cells.
The light and truncated heavy chains of human factor VIII, expressed separately in baculovirus-infected insect cells, exhibited different secretory behaviour when compared with each other and with a biologically active fusion molecule of the truncated heavy and light chains. The light chain was very efficiently secreted into culture medium, as judged by high extracellular protein levels and the absence of evidence for light chain retention within cells. Alternatively, proteins containing the heavy chain sequence were poorly secreted and appeared to be sequestered within cells, suggesting that regions within the heavy chain are responsible for the low levels of secreted protein which have generally been observed for recombinant factor VIII.
Animals↗
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.
Animals↗