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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

12 Issues per year


IMPACT FACTOR 2016: 3.273

CiteScore 2016: 3.01

SCImago Journal Rank (SJR) 2016: 1.679
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1437-4315
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Volume 386, Issue 8 (Aug 2005)

Issues

Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC

Markus J. Stemp
  • Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany
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/ Suranjana Guha
  • Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany
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/ F. Ulrich Hartl
  • Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany
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/ José M. Barral
  • Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany
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Published Online: 2005-09-06 | DOI: https://doi.org/10.1515/BC.2005.088

Abstract

Recombinant expression of actin in bacteria results in non-native species that aggregate into inclusion bodies. Actin is a folding substrate of TRiC, the chaperonin of the eukaryotic cytosol. By employing bacterial in vitro translation lysates supplemented with purified chaperones, we have found that TRiC is the only eukaryotic chaperone necessary for correct folding of newly translated actin. The actin thus produced binds deoxyribonuclease I and polymerizes into filaments, hallmarks of its native state. In contrast to its rapid folding in the eukaryotic cytosol, actin translated in TRiC-supplemented bacterial lysate folds with slower kinetics, resembling the kinetics upon refolding from denaturant. Lysate supplementation with the bacterial chaperonin GroEL/ES or the DnaK/DnaJ/GrpE chaperones leads to prevention of actin aggregation, yet fails to support its correct folding. This combination of in vitro bacterial translation and TRiC-assisted folding allows a detailed analysis of the mechanisms necessary for efficient actin folding in vivo. In addition, it provides a robust alternative for the production of substantial amounts of eukaryotic proteins that otherwise misfold or lead to cellular toxicity upon expression in heterologous hosts.

Keywords: molecular chaperones; protein folding; recombinant expression

About the article

Corresponding author


Received: March 16, 2005

Accepted: April 7, 2005

Published Online: 2005-09-06

Published in Print: 2005-08-01


Citation Information: Biological Chemistry, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2005.088.

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