Expression of P-450 Enzyme Activities in Heterologous Cells by Transfection
The product of the A gene of simian virus 40 (SV40), T antigen, is synthesized and accumulates in the nucleus of infected cells. This regulartory protein acts to initiate viral DNA replication as well as regulating SV40 gene transcription (Tjian 1981). Following transformation of CV-1 cells with an origin-defective mutant of SV40, the resultant monkey kidney cell line (called COS 1) produces T antigen (Gluzman 1981). Transfection of COS 1 cells with plasmid vectors which contain an SV40 origin of replication leads to replication of the plasmid DNA under the influence of T antigen. Accordingly the number of plasmid vectors is amplified in transfected COS 1 cells and the amplified plasmid DNA can then be transcribed leading to production of relatively large quantities of RNA derived from the plasmid. This RNA can, of course, be translated by the endogenous protein synthetic machinery in the COS 1 cells. When the plasmid vector contains a cDNA insert encoding a specific protein, readily detectable quantities of the protein encoded by the insert can be produced in COS 1 cells. A convenient vector for such expression studies is the pcD vector constructed by Okayama and Berg (1982) which contains an SV40 origin of replication and SV40 promotor sequence. A similar vector (pSVL) can be purchased from Pharmacia. pSVL contains a limited multiple cloning site which facilitates insertion of different cDNAs into this expression vector.
KeywordsHeterologous Cell Steroidogenic Pathway Steroid Hydroxylase Bovine Enzyme Adrenodoxin Reductase
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