Journal
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
Volume 59, Issue 4, Pages 314-321Publisher
WILEY
DOI: 10.1002/bab.1029
Keywords
baculovirus; insect cell; ligand binding; receptor signaling; receptor expression; RSV promoter
Funding
- Academy of Finland
- Sigrid Juselius Foundation
- Magnus Ehrnrooth Foundation
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The effect of the Rous sarcoma virus (RSV) long terminal repeat enhancer/promoter on expression levels of complementary DNAs (cDNAs) encoding seven transmembrane receptors was studied using the baculovirus expression vector system. Expression of the human a2B-adrenoceptor (AR) cDNA under the control of the polyhedrin (POL) promoter produced up to 7.6?pmol/mg protein at 28 H post infection (p.i.) in Sf9 cells. The addition of the RSV promoter increased the expression to 11.6?pmol/mg protein. Dramatic increases in expression levels at early times were also obtained with the a2A-AR, the M1 and M4 muscarinic receptors, and the orexin OX1 receptor. Analysis of the time-dependent expression revealed that expression driven by the RSV promoter reaches almost maximum 24?H p.i. and that this promoter is superior to the often used POL promoter at early times p.i. when functional studies need to be performed. Functional enhancement of signaling as a result of early expression is demonstrated with the a2B-AR and the OX1 receptor. Finally, enhanced green fluorescent protein fluorescence in living cells was used to monitor expression by various viral promoters. The results verified the early transcriptional activity of the RSV promoter, whereas the cytomegalovirus promoter was found to be poorly active in Sf9 cells.
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