title: Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.
abstract: Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.
authors.affiliation: European Molecular Biology Laboratory (EMBL), Grenoble Outstation, and Unit of Virus Host Cell Interactions UVHCI, UJF-CNRS-EMBL Unite Mixte International UMI 3265, Rue Jules Horowitz, 38042 Grenoble Cedex 9, France.
authors.forename: Lakshmi Sumitra
authors.initials: LS
authors.lastname: Vijayachandran
authors.forename: Cristina
authors.initials: C
authors.lastname: Viola
authors.forename: Frederic
authors.initials: F
authors.lastname: Garzoni
authors.forename: Simon
authors.initials: S
authors.lastname: Trowitzsch
authors.forename: Christoph
authors.initials: C
authors.lastname: Bieniossek
authors.forename: Maxime
authors.initials: M
authors.lastname: Chaillet
authors.forename: Christiane
authors.initials: C
authors.lastname: Schaffitzel
authors.forename: Didier
authors.initials: D
authors.lastname: Busso
authors.forename: Christophe
authors.initials: C
authors.lastname: Romier
authors.forename: Arnaud
authors.initials: A
authors.lastname: Poterszman
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Richmond
authors.forename: Imre
authors.initials: I
authors.lastname: Berger
mesh_terms: D000047:Academies and Institutes; D000818:Animals; D057205:Automation, Laboratory / Q000295:instrumentation*; D016367:Baculoviridae; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D005060:Europe; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis; D046912:Multiprotein Complexes / Q000096:biosynthesis*; D020815:Polyproteins / Q000096:biosynthesis / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera
chemical_list: D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D020815:Polyproteins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins