Abstract
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.
Copyright © 2011 Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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CCAAT-Binding Factor / biosynthesis
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CCAAT-Binding Factor / chemistry
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CCAAT-Binding Factor / genetics
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Cloning, Molecular / methods*
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Drosophila
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Drosophila Proteins / biosynthesis
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Drosophila Proteins / chemistry
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Drosophila Proteins / genetics
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Escherichia coli / genetics
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Genetic Vectors
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Histones / biosynthesis
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Histones / genetics*
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Humans
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Multiprotein Complexes / biosynthesis
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Multiprotein Complexes / chemistry
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Multiprotein Complexes / genetics*
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Nuclear Proteins / biosynthesis
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nucleosomes / genetics
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Nucleosomes / metabolism
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RNA-Binding Proteins / biosynthesis
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RNA-Binding Proteins / chemistry
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RNA-Binding Proteins / genetics
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics*
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Saccharomyces cerevisiae
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Saccharomyces cerevisiae Proteins / biosynthesis
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Trans-Activators / biosynthesis
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Trans-Activators / chemistry
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Trans-Activators / genetics
Substances
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CCAAT-Binding Factor
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Drosophila Proteins
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Histones
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Mago protein, Drosophila
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Multiprotein Complexes
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NFYA protein, human
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NFYB protein, human
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NFYC protein, human
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Nuclear Proteins
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Nucleosomes
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PYM protein, Drosophila
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RNA-Binding Proteins
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Recombinant Proteins
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SAGA complex, S cerevisiae
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Saccharomyces cerevisiae Proteins
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Trans-Activators
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tsu protein, Drosophila