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.

Activities of the chaperonin containing TCP-1 (CCT): implications for cell cycle progression and cytoskeletal organisation.

“…This theory is further supported by the work of Stemp et al (2005) and Pappenberger et al (2006) who both show that a stablefolding intermediate of actin formed in an Escherichia coli lysate in vitro translation system can reach its native form when in the presence of CCT. Furthermore, it is shown that CCT is necessary for the correct folding of newly synthesized actin and that the GroEL system cannot replace this activity (Stemp et al 2005).…”

A new bioproduction route for a novel antimicrobial peptide

“…It is important to recognize that the production of soluble protein does not always guarantee monomericity or bioactivity of the target protein (Stemp et al, 2005;Yee et al, 2001). To determine the monomericity of the His-MBP-hBD28 fusion protein, the sonicated cell lysates were purified to remove host contaminants and loaded into a SEC column for soluble aggregate analysis.…”

Quantitative Actin Folding Reactions using Yeast CCT Purified via an Internal Tag in the CCT3/γ Subunit

“…The E. coli extract is a total cleared cell lysate and thus contains bacterial chaperones such as GroEL/ES or DnaK/DnaJ/ GrpE, and some of these components may assist the prefolding of nascent actin chains and/or stabilise the Ac I intermediate once it is partially folded. 42 For example, although the Ac I chains migrate discretely on native gels, they are highly prone to aggregation; heating them to 46°C for a few minutes elicits complete aggregation compared to native actin, which is very much more stable ( Figure 6(a) and (b)). It will now be possible to ask whether any bacterial chaperones are able to enhance Ac I stability by preventing aggregation and whether any can increase native actin yield in the pure yeast CCT system.…”