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

Biol Chem. 2005 Aug;386(8):753-7. doi: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / genetics
  • Actins / metabolism*
  • Bacteria / metabolism*
  • Cell Extracts
  • Chaperonin 10 / genetics
  • Chaperonin 10 / metabolism
  • Chaperonin Containing TCP-1
  • Chaperonins / genetics
  • Chaperonins / metabolism*
  • Cytosol / metabolism
  • Deoxyribonuclease I / genetics
  • Deoxyribonuclease I / metabolism
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Eukaryotic Cells / metabolism*
  • HSP40 Heat-Shock Proteins / genetics
  • HSP40 Heat-Shock Proteins / metabolism
  • HSP70 Heat-Shock Proteins / genetics
  • HSP70 Heat-Shock Proteins / metabolism
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism
  • Kinetics
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Protein Biosynthesis*
  • Protein Denaturation
  • Protein Folding
  • Proteins / genetics
  • Proteins / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • t-Complex Genome Region

Substances

  • Actins
  • Broncho-Vaxom
  • Cell Extracts
  • Chaperonin 10
  • DnaJ protein, E coli
  • Escherichia coli Proteins
  • GrpE protein, E coli
  • HSP40 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Microtubule-Associated Proteins
  • Nuclear Proteins
  • Proteins
  • Recombinant Proteins
  • Deoxyribonuclease I
  • Chaperonin Containing TCP-1
  • Chaperonins
  • dnaK protein, E coli