Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC
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Markus J. Stemp
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.
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©2005 by Walter de Gruyter Berlin New York
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Articles in the same Issue
- Highlight: Molecular Machines
- Splicing of a rare class of introns by the U12-dependent spliceosome
- Proteasome-associated proteins: regulation of a proteolytic machine
- Novel insights into the mechanism of chaperone-assisted protein disaggregation
- Dynamic chromatin: concerted nucleosome remodelling and acetylation
- Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC
- The NC1 dimer of human placental basement membrane collagen IV: does a covalent crosslink exist?
- Intact growth factors are conserved in the extracellular matrix of ancient human bone and teeth: a storehouse for the study of human evolution in health and disease
- Differential accumulation of plastid preprotein translocon components during spruce (Picea abies L. Karst.) needle development
- Hypothyroidism induces expression of the peptide transporter PEPT2
- The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase
- Effects of elastase and cathepsin G on the levels of membrane and soluble TNFα
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