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

Markus J. Stemp, Suranjana Guha, F. Ulrich Hartl, José M. Barral

Research output: Contribution to journalArticle

15 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)753-757
Number of pages5
JournalBiological Chemistry
Volume386
Issue number8
DOIs
StatePublished - 2005
Externally publishedYes

Fingerprint

Chaperonins
Actins
Cytosol
Kinetics
Deoxyribonuclease I
Inclusion Bodies
Broncho-Vaxom
Toxicity
Bacteria
Agglomeration
Substrates

Keywords

  • Molecular chaperones
  • Protein folding
  • Recombinant expression

ASJC Scopus subject areas

  • Biochemistry

Cite this

Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC. / Stemp, Markus J.; Guha, Suranjana; Hartl, F. Ulrich; Barral, José M.

In: Biological Chemistry, Vol. 386, No. 8, 2005, p. 753-757.

Research output: Contribution to journalArticle

Stemp, Markus J. ; Guha, Suranjana ; Hartl, F. Ulrich ; Barral, José M. / Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC. In: Biological Chemistry. 2005 ; Vol. 386, No. 8. pp. 753-757.
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