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  Comparative analysis and "expression space" coverage of the production of prokaryotic membrane proteins for structural genomics

Surade, S., Klein, M., Stolt-Bergner, P. C., Muenke, C., Roy, A., & Michel, H. (2006). Comparative analysis and "expression space" coverage of the production of prokaryotic membrane proteins for structural genomics. Protein Science, 15(9), 2178-2189. doi:10.1110/ps.062312706.

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 Creators:
Surade, Sachin1, Author              
Klein, Markus1, Author              
Stolt-Bergner, Peggy C.1, Author              
Muenke, Cornelia1, Author              
Roy, Ankita1, Author              
Michel, Hartmut1, Author              
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

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Free keywords: membrane proteins; transporters; structural genomics; protein production; high-throughput screen
 Abstract: Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.

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Language(s): eng - English
 Dates: 2006-06-232006-04-272006-06-232006-09-01
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1110/ps.062312706
PMID: 16943447
 Degree: -

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Title: Protein Science
Source Genre: Journal
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Publ. Info: New York, N.Y. : Cambridge University Press
Pages: - Volume / Issue: 15 (9) Sequence Number: - Start / End Page: 2178 - 2189 Identifier: ISSN: 0961-8368
CoNE: https://pure.mpg.de/cone/journals/resource/954925342760