Expression of cell surface GPI-anchored human p97 in baculovirus-infected insect cells.
The baculovirus/insect cell system (Autographa californica multiple nuclear polyhedrosis virus/Spodoptera frugiperda Sf9 cells) was used to express the GPI-anchored human melanoma tumor antigen, melanotransferrin or p97. This system served to study the expression and productivity of recombinant GPI-anchored p97 by insect cells. The Sf9 cells expressed a cell surface GPI-anchored form of p97 as well as a soluble form of p97 that did not appear to be derived from the GPI-anchored form of p97. Both recombinant forms, although Endo H resistant, migrated slightly faster ( approximately 88 kDa) than the native p97 ( approximately 95-97 kDa). The insect GPI-anchored p97 was sensitive to PI-PLC, which exposed a detectable cross-reacting determinant. The Sf9 cell surface p97 expression was similar to that of human melanoma (SK-MEL-28) cells, whereas the Sf9 cell specific secretion rate was 10-fold higher. Also Sf9 cells retained considerably higher levels of p97 within the cell. The Sf9 cell surface expression of p97 varied with time after infection, with the maximum expression, which appeared independent of multiplicities of infection greater than 1, occurring at 48 h. After 48 h, levels of cell surface and secreted p97 fell whereas p97 retained within the cell increased, which possibly reflected the lytic nature of the expression system. The successful expression of GPI-anchored human p97 by the baculovirus/insect cell system not only provides a source of p97 for further research but also is the basis of an alternative method for the commercial production of GPI-anchored proteins.
Kennard
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Biotechnology Laboratory and Departments of Medical Genetics, Microbiology and Zoology, University of British Columbia, Room 237 Wesbrook Building, 6174 University Boulevard, Vancouver, British Columbia, Canada.
Shimizu
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Gabathuler
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Rothenberger
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Theilmann
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Jefferies
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