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- Title
Enhanced early expression of membrane receptors with the Rous sarcoma virus promoter in baculovirus-infected insect cells.
- Authors
Näreoja, Katja; Åkerman, Karl E.; Näsman, Johnny
- Abstract
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 α2B-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 α2A-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 α2B-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.
- Subjects
ROUS sarcoma; BACULOVIRUS diseases; PROMOTERS (Genetics); ADRENERGIC receptors; MUSCARINIC receptors; GENE expression
- Publication
Biotechnology & Applied Biochemistry, 2012, Vol 59, Issue 4, p314
- ISSN
0885-4513
- Publication type
Article
- DOI
10.1002/bab.1029