Authors list:
Frederic Garzoni, Christoph Bieniossek, Imre Berger

Abstract:

Protein complexes are central to cellular function. Many important complexes in eukaryotes require recombinant overproduction for a detailed analysis of their structure and function. Baculovirus expression vector systems (BEVS) have become increasingly popular for the production of such specimens, in particular for complexes which depend on a eukaryotic host cell machinery for proper folding, post-translational modification, authentic processing and correct targeting to cell compartments for their activity. The MultiBac system (Fig. 1) is a BEVS specifically designed for producing large eukaryotic complexes with many subunits [Berger et al., Nat. Biotechnol. 2004, Fitzgerald et al., Nat Methods, 2006]. It consists of an array of plasmids which facilitate multigene assembly, a modified baculovirus genome that with optimized protein production properties, and a set of protocols detailing every step from inserting encoding DNAs in the plasmid array to protein production by this technology. The components of the system and the protocols used are continuously being improved, developed and streamlined to simplify handling and improve efficacy [Trowitzsch et al., J. Struct. Biol. 2010, Vijayachandran et al., J. Struct. Biol. 2011]. We believe that our efforts have reduced the previously perceived complexity of the baculovirus/insect cell system to the level of protein expression in E. coli. Detailed protocols are available for utilizing the MultiBac system [Fitzgerald et al., Nat. Methods 2006; Bieniossek et al., Curr. Protoc. Prot. Sci. 2008], although certain details have been altered over the years and are bound to be refined in the future. In this protocol, we present the protocol as it is currently in operation at the MultiBac platform of Eukaryotic Expression Facility (EEF) at the EMBL Grenoble (Fig. 2), as part of the trans-national access scheme in the P-CUBE (www.p-cube.eu) and BioSTRUCT-X (www.biostruct-x.eu) projects of the European Commission, Framework Program 7. The protocol has been designed such that it can be performed successfully by scientists with or without previous knowledge of or experience with BEVS for expressing their targets during a ten working-day visit at our MultiBac platform.Parts of the protocols are specific for MultiBac (plasmid fusions, multigene generation, fluorescence tracking). However, all virus amplification and protein production protocols are generically applicable to other baculovirus systems that utilize recombinant bacmids (for example Bac-to-Bac, Invitrogen).