Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Protein Science (2006), 15:1433-1440. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chelikani, P.
Right arrow Articles by Khorana, H. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chelikani, P.
Right arrow Articles by Khorana, H. G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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

Prashen Chelikani1,2, Philip J. Reeves1,2,3, Uttam L. Rajbhandary1 and H. Gobind Khorana1,2

Departments of 1 Biology
2 Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

(RECEIVED January 5, 2006; FINAL REVISION March 14, 2006; ACCEPTED March 14, 2006)

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 µg/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 µg/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.

Keywords: GPCR; synthetic gene; beta-adrenergic receptor; expression; mammalian cell lines


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
P. Chelikani, V. Hornak, M. Eilers, P. J. Reeves, S. O. Smith, U. L. RajBhandary, and H. G. Khorana
Role of group-conserved residues in the helical core of beta2-adrenergic receptor
PNAS, April 24, 2007; 104(17): 7027 - 7032.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2006 by The Protein Society.