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  Cell-Free Expression and Assembly of ATP Synthase

Matthies, D., Haberstock, S., Joos, F., Dötsch, V., Vonck, J., Bernhard, F., et al. (2011). Cell-Free Expression and Assembly of ATP Synthase. Journal of Molecular Biology, 413, 593-603. doi:10.1016/j.jmb.2011.08.055.

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 Creators:
Matthies, Doreen1, Author           
Haberstock, Stefan2, Author
Joos, Friederike1, Author           
Dötsch, Volker2, 3, Author
Vonck, Janet1, Author           
Bernhard, Frank2, Author
Meier, Thomas1, 3, Author           
Affiliations:
1Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068291              
2Center for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University Frankfurt, 60438 Frankfurt am Main, Germany, ou_persistent22              
3Cluster of Excellence Macromolecular Complexes, Frankfurt am Main, Germany, ou_persistent22              

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Free keywords: cell-free expression; in vitro protein synthesis and assembly; F1Fo-ATP synthase; membrane protein complex; Caldalkalibacillus thermarum strain TA2.A1
 Abstract: 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 F1Fo-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry α3β3γδɛab2c13. 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.

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Language(s): eng - English
 Dates: 2011-08-112011-06-232011-08-302011-09-102011-10-28
 Publication Status: Published in print
 Pages: 11
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.jmb.2011.08.055
PMID: 21925509
 Degree: -

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Title: Journal of Molecular Biology
  Other : JMB
  Abbreviation : J. Mol. Biol.
Source Genre: Journal
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Publ. Info: Elsevier
Pages: - Volume / Issue: 413 Sequence Number: - Start / End Page: 593 - 603 Identifier: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/0022-2836