Purification and functional properties of simian virus 40 large and small T antigens overproduced in insect cells.
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.
Murphy
C I
CI
Dana-Farber Cancer Institute, Boston, Massachusetts.
Weiner
B
B
Bikel
I
I
Piwnica-Worms
H
H
Bradley
M K
MK
Livingston
D M
DM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000302:isolation & purification*; D001681:Biological Assay; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D007526:Isoelectric Point; D008970:Molecular Weight; D011994:Recombinant Proteins; D012995:Solubility
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases