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Published online before print July 1, 2008, 10.1110/ps.035980.108
Protein Science (2008), 17:1857-1863. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 The Protein Society
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FOR THE RECORD

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli

A. James Link1,2,6, Georgios Skretas1,2, Eva-Maria Strauch3, Nandini S. Chari3, and George Georgiou1,2,4,5

1 Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA
2 Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA
3 Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, USA
4 Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA
5 Department of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas 78712, USA

(RECEIVED April 24, 2008; FINAL REVISION June 19, 2008; ACCEPTED June 20, 2008)

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.

Keywords: G protein-coupled receptor; Escherichia coli; membrane protein; FtsH; genetic engineering; cannabinoid receptor; bradykinin receptor


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