title: Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer.
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.
authors.affiliation: 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.
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Diebold
authors.forename: Sébastien
authors.initials: S
authors.lastname: Fribourg
authors.forename: Michael
authors.initials: M
authors.lastname: Koch
authors.forename: Thibaud
authors.initials: T
authors.lastname: Metzger
authors.forename: Christophe
authors.initials: C
authors.lastname: Romier
mesh_terms: D000818:Animals; D023081:CCAAT-Binding Factor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004330:Drosophila; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006657:Histones / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D046912:Multiprotein Complexes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015534:Trans-Activators / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D023081:CCAAT-Binding Factor; D029721:Drosophila Proteins; D006657:Histones; C107372:Mago protein, Drosophila; D046912:Multiprotein Complexes; C492061:NFYA protein, human; C492063:NFYB protein, human; C469358:NFYC protein, human; D009687:Nuclear Proteins; D009707:Nucleosomes; C493333:PYM protein, Drosophila; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C507785:SAGA complex, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C440016:tsu protein, Drosophila