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 ~1 to 2 μg of t and up to 30 μg of T per 3 x 106 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.
Original language | English (US) |
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Pages (from-to) | 2951-2959 |
Number of pages | 9 |
Journal | Journal of Virology |
Volume | 62 |
Issue number | 8 |
DOIs | |
State | Published - 1988 |
Externally published | Yes |
ASJC Scopus subject areas
- Microbiology
- Immunology
- Insect Science
- Virology