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Purification and functional properties of simian virus 40 large and small T antigens overproduced in insect cells.

By C I Murphy, B Weiner, I Bikel, H Piwnica-Worms, M K Bradley and D M Livingston

Abstract

The insect baculovirus Autographa californica nuclear polyhedrosis virus was used as an expression vector for the simian virus 40 (SV40) small t (t) and large T (T) antigens. Spodoptera frugiperda (SF9) cells infected with recombinant viruses encoding these proteins produced approximately 1 to 2 micrograms of t and up to 30 micrograms of T per 3 X 10(6) cells. The former was highly soluble after Nonidet P-40 extraction of the infected cells, unlike its Escherichia coli-produced counterpart. Both SF9-produced proteins were of authentic size and could be readily immunoprecipitated by specific antibodies. Single-step immunoaffinity chromatography was used to purify the two proteins to near homogeneity, with yields averaging 70% in each case. Experiments to test the biological activity of the baculovirus SV40 proteins showed that SF9 t was capable of associating with two of the cellular proteins reported to bind to t in SV40-infected mammalian cells. Moreover, SF9 T had ATPase activity comparable to that of T produced in monkey cells, exhibited helicase activity and SV40 origin-specific DNA binding, and was active in the SV40 DNA replication assay in vitro. Thus, the SV40 T antigens produced in insect cells can be used in future studies of their biochemical roles in vitro and in vivo

Topics: Research Article
Year: 1988
OAI identifier: oai:pubmedcentral.nih.gov:253733
Provided by: PubMed Central
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