Back to Search Start Over

The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line.

Authors :
Chelikani P
Reeves PJ
Rajbhandary UL
Khorana HG
Source :
Protein science : a publication of the Protein Society [Protein Sci] 2006 Jun; Vol. 15 (6), pp. 1433-40.
Publication Year :
2006

Abstract

High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta2-adrenergic receptor (beta2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta2-AR/mg of membrane protein, as measured by saturation binding assay using the beta2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta2-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 microg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 microg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta2-AR expression using suspension cultures in a bioreactor should now allow production of enough beta2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.

Details

Language :
English
ISSN :
0961-8368
Volume :
15
Issue :
6
Database :
MEDLINE
Journal :
Protein science : a publication of the Protein Society
Publication Type :
Academic Journal
Accession number :
16731977
Full Text :
https://doi.org/10.1110/ps.062080006