Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer

Marie-Laure Diebold; Sébastien Fribourg; Michael Koch; Thibaud Metzger; Christophe Romier

doi:10.1016/j.jsb.2011.02.001

Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer

J Struct Biol. 2011 Aug;175(2):178-88. doi: 10.1016/j.jsb.2011.02.001. Epub 2011 Feb 12.

Authors

Marie-Laure Diebold¹, Sébastien Fribourg, Michael Koch, Thibaud Metzger, Christophe Romier

Affiliation

¹ Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie Intégrative, Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 1704/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch, France.

PMID: 21320604
DOI: 10.1016/j.jsb.2011.02.001

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CCAAT-Binding Factor / biosynthesis
CCAAT-Binding Factor / chemistry
CCAAT-Binding Factor / genetics
Cloning, Molecular / methods*
Drosophila
Drosophila Proteins / biosynthesis
Drosophila Proteins / chemistry
Drosophila Proteins / genetics
Escherichia coli / genetics
Genetic Vectors
Histones / biosynthesis
Histones / genetics*
Humans
Multiprotein Complexes / biosynthesis
Multiprotein Complexes / chemistry
Multiprotein Complexes / genetics*
Nuclear Proteins / biosynthesis
Nuclear Proteins / chemistry
Nuclear Proteins / genetics
Nucleosomes / genetics
Nucleosomes / metabolism
RNA-Binding Proteins / biosynthesis
RNA-Binding Proteins / chemistry
RNA-Binding Proteins / genetics
Recombinant Proteins / biosynthesis
Recombinant Proteins / chemistry
Recombinant Proteins / genetics*
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins / biosynthesis
Saccharomyces cerevisiae Proteins / chemistry
Saccharomyces cerevisiae Proteins / genetics
Trans-Activators / biosynthesis
Trans-Activators / chemistry
Trans-Activators / genetics

Substances

CCAAT-Binding Factor
Drosophila Proteins
Histones
Mago protein, Drosophila
Multiprotein Complexes
NFYA protein, human
NFYB protein, human
NFYC protein, human
Nuclear Proteins
Nucleosomes
PYM protein, Drosophila
RNA-Binding Proteins
Recombinant Proteins
SAGA complex, S cerevisiae
Saccharomyces cerevisiae Proteins
Trans-Activators
tsu protein, Drosophila