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

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.

journal_name

Biol Chem

journal_title

Biological chemistry

authors

Stemp MJ,Guha S,Hartl FU,Barral JM

doi

10.1515/BC.2005.088

keywords:

subject

Has Abstract

pub_date

2005-08-01 00:00:00

pages

753-7

issue

8

eissn

1431-6730

issn

1437-4315

journal_volume

386

pub_type

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