Quick Search:

uzh logo
Browse by:
bullet
bullet
bullet
bullet

Zurich Open Repository and Archive 

Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-4809

Sarkar, C A; Dodevski, I; Kenig, M; Dudli, S; Mohr, A; Hermans, E; Plückthun, A (2008). Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proceedings of the National Academy of Sciences of the United States of America (PNAS), 105(39):14808-14813.

[img] PDF - Registered users only
1MB

Abstract

We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
DDC:570 Life sciences; biology
Language:English
Date:30 September 2008
Deposited On:29 Oct 2008 16:13
Last Modified:27 Nov 2013 17:09
Publisher:National Academy of Sciences
ISSN:0027-8424
Additional Information:Copyright: National Academy of Sciences USA
Publisher DOI:10.1073/pnas.0803103105
Official URL:http://www.pnas.org/content/105/39/14808
Related URLs:http://www.pnas.org/content/105/39/14808/F2.expansion.html
PubMed ID:18812512
Citations:Web of Science®. Times Cited: 64
Google Scholar™
Scopus®. Citation Count: 66

Users (please log in): suggest update or correction for this item

Repository Staff Only: item control page