UZH-Logo

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity


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.

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.

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.

Citations

90 citations in Web of Science®
89 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

2 downloads since deposited on 29 Oct 2008
0 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:30 September 2008
Deposited On:29 Oct 2008 16:13
Last Modified:05 Apr 2016 12:31
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
Permanent URL: http://doi.org/10.5167/uzh-4809

Download

[img]
Filetype: PDF - Registered users only
Size: 1MB
View at publisher

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations