|FoldHelper™-104 and FoldHelper™-104P||Cat. #H15 and #H16|
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).
| Recommended use:
Co-infection with any recombinant baculoviruses producing cytoplasmic
|FoldHelper™-104 provides for expression of yeast Hsp104 (Acc# AAA50477). Hsp104 belongs to Hsp100/Clp group of molecular chaperones, which can be used as tools for reversing, rather than preventing protein aggregation (Glover J.R. and Tkach, J.M., Biochem. Cell Biol., 79: 557-568, 2001). Hsp104 has remodeling activity that acts on trapped, aggregated proteins. Hsp104 requires specific interaction with conventional chaperones, especially belonging to Hsp40 and Hsp70 families, to promote refolding of the intermediates it produces (Glover J.R. and Lindquist S., Cell, 94: 73-82, 1998). Yeast Hsp104 can function in heterologous systems, e.g., it can reduce aggregation of foreign proteins in mammalian cells (Bao Y.P. et al., J. Biol. Chem., 277: 12263-12269, 2002).
FoldHelper™-104P provides for the same level of expression of Hsp104 as FoldHelper™-104. In addition, it provides for high-level expression of protein disulfide isomerase (PDI), (Acc# CAA28775). PDI was demonstrated to be efficient in oxidative protein folding in vitro (Weissman, J.S. and Kim P.S., Nature, 365: 185-188, 1993). PDI 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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