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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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Matthies, Doreen1, Autor           
Haberstock, Stefan2, Autor
Joos, Friederike1, Autor           
Dötsch, Volker2, 3, Autor
Vonck, Janet1, Autor           
Bernhard, Frank2, Autor
Meier, Thomas1, 3, Autor           
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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Schlagwörter: cell-free expression; in vitro protein synthesis and assembly; F1Fo-ATP synthase; membrane protein complex; Caldalkalibacillus thermarum strain TA2.A1
 Zusammenfassung: 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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Sprache(n): eng - English
 Datum: 2011-08-112011-06-232011-08-302011-09-102011-10-28
 Publikationsstatus: Erschienen
 Seiten: 11
 Ort, Verlag, Ausgabe: -
 Inhaltsverzeichnis: -
 Art der Begutachtung: Expertenbegutachtung
 Identifikatoren: DOI: 10.1016/j.jmb.2011.08.055
PMID: 21925509
 Art des Abschluß: -

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Titel: Journal of Molecular Biology
  Andere : JMB
  Kurztitel : J. Mol. Biol.
Genre der Quelle: Zeitschrift
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Affiliations:
Ort, Verlag, Ausgabe: Elsevier
Seiten: - Band / Heft: 413 Artikelnummer: - Start- / Endseite: 593 - 603 Identifikator: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/0022-2836