Spodoptera frigiperda Sf9 cells were harvested at 60 h post infection, lysed in 50 mM Tris-HCl pH 8.0, 150 mM EDTA, 0.5% NP-40, and insoluble fraction was removed by centrifugation at 30.000g for 15 min. Soluble proteins were separated in 11% SDS-PAGE and stained with Coomassie blue. NC-negative control baculovirus, which does not express any foreign proteins or polyhedrin ( Cat. #C13 ).
Co-infection with any recombinant baculoviruses producing proteins having multiple cysteines.
|FoldHelper™-57P provides for expression of human ERp57 (Acc# NP_005304) and human protein disulfide isomerase (PDI) (Acc# CAA28775.1). ERp57 and PDI are homologous proteins participating in disulfide bond formation and oxidative protein folding in the endoplasmic reticulum (Frand A.R.et al., Trends in Cell Biol., 10: 203-210, 2000). PDI was demonstrated to be efficient in oxidative protein folding in vitro (Weissman, J.S. and Kim P.S., Nature, 365: 185-188, 1993). PDIs have multiple functions and, though most abundant in the ER, it is also found in the cytoplasm (Turano, C., et al., J. Cell. Physiol., 193: 154-163, 2002).
A welcome, albeit unexplained effect of PDI in insect cells infected with recombinant baculoviruses, is that PDI improves longevity of such cells. This cytopathic effect is markedly less pronounced in infected cells expressing a high level of PDI, and such cells survive about 24 hours longer than cells infected with a control virus which does not provide for PDI expression. Similarly, PDI overexpression in mammalian cells resulted in increased longevity and time of recombinant protein production (Kitchin, K and Flickinger, M.C., Biotechnol. Progr., 11: 565-574, 1995).
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