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Cell-Free Expression and Assembly of ATP Synthase.
MedLine Citation:
PMID:  21925509     Owner:  NLM     Status:  Publisher    
Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the  542-kDa F(1)F(o)-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry α(3)β(3)γδɛab(2)c(13). Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems.
Doreen Matthies; Stefan Haberstock; Friederike Joos; Volker Dötsch; Janet Vonck; Frank Bernhard; Thomas Meier
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-9-10
Journal Detail:
Title:  Journal of molecular biology     Volume:  -     ISSN:  1089-8638     ISO Abbreviation:  -     Publication Date:  2011 Sep 
Date Detail:
Created Date:  2011-9-19     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  2985088R     Medline TA:  J Mol Biol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2011 Elsevier Ltd. All rights reserved.
Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt/Main, Germany.
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