Macromolecular complexes are responsible for most of the essential mechanisms in cells, leading to a broad interest in their purification and characterization. Co-expression is now widely recognized as a major technique for assembling multiprotein complexes and many co-expression systems are currently available for performing co-expression experiments in different hosts. However, comparative knowledge on co-expression strategies is still crucially lacking. Using versatile co-expression systems for Escherichia coli, the pET-MCN and pET-MCP series, and ternary protein complexes as examples, we demonstrate how to successfully delineate correct co-expression strategies. Specifically, an appropriate, complex-dependent approach alleviates stoichiometry imbalance and yield problems, and even failure in producing complexes. Importantly, some of the parameters influencing co-expression strategies appear independent of the expression host, thus having implications for co-expression in eukaryotic hosts. By further using these strategies, we show that co-expression in E. coli enables reconstitution of protein complexes as large as the deubiquitination module of the SAGA transcription factor and the histone octamer.

• PMID: 21320604
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Diebold ML, Fribourg S, Koch M, Metzger T, Romier C

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