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.
Dates
Type | When |
---|---|
Created | 5 years, 7 months ago (Jan. 6, 2020, 9:58 a.m.) |
Deposited | 3 years, 5 months ago (March 4, 2022, 6:26 p.m.) |
Indexed | 1 year, 10 months ago (Oct. 1, 2023, 8:49 a.m.) |
Issued | 37 years ago (Aug. 1, 1988) |
Published | 37 years ago (Aug. 1, 1988) |
Published Print | 37 years ago (Aug. 1, 1988) |
@article{Murphy_1988, title={Purification and functional properties of simian virus 40 large and small T antigens overproduced in insect cells}, volume={62}, ISSN={1098-5514}, url={http://dx.doi.org/10.1128/jvi.62.8.2951-2959.1988}, DOI={10.1128/jvi.62.8.2951-2959.1988}, number={8}, journal={Journal of Virology}, publisher={American Society for Microbiology}, author={Murphy, C I and Weiner, B and Bikel, I and Piwnica-Worms, H and Bradley, M K and Livingston, D M}, year={1988}, month=aug, pages={2951–2959} }