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


     

Protein Science (2007), 16:966-976. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
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 Savage, D. F.
Right arrow Articles by Stroud, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Savage, D. F.
Right arrow Articles by Stroud, R. M.
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?

Cell-free complements in vivo expression of the E. coli membrane proteome

David F. Savage1,4, Corey L. Anderson3,4, Yaneth Robles-Colmenares3, Zachary E. Newby2, and Robert M. Stroud3

1 Graduate Group in Biophysics, University of California at San Francisco, San Francisco, California 94158-2517, USA
2 Chemistry and Chemical Biology Graduate Program, University of California at San Francisco, San Francisco, California 94158-2517, USA
3 Centre for Structure of Membrane Proteins, University of California at San Francisco, San Francisco, California 94158-2517, USA

(RECEIVED November 29, 2006; FINAL REVISION January 11, 2007; ACCEPTED January 11, 2007)

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.

Keywords: cell-free protein expression; integral membrane proteins; structural genomics; high-throughput protein expression


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
L. Kaiser, J. Graveland-Bikker, D. Steuerwald, M. Vanberghem, K. Herlihy, and S. Zhang
Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses
PNAS, October 14, 2008; 105(41): 15726 - 15731.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2007 by The Protein Society.