English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

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

MPS-Authors
/persons/resource/persons137912

Surade,  Sachin
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137744

Klein,  Markus
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137908

Stolt-Bergner,  Peggy C.
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137814

Muenke,  Cornelia
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137859

Roy,  Ankita
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137800

Michel,  Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

External Ressource
No external resources are shared
Fulltext (public)
There are no public fulltexts stored in PuRe
Supplementary Material (public)
There is no public supplementary material available
Citation

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.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-D9BF-D
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.