The Production of Multiprotein Complexes in Insect Cells Using the Baculovirus Expression System
The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.
Key wordsRecombinant protein complex production BEVS Baculovirus Insect cells Infection and coinfection methods Multigene expression Multiprotein complex
We thank Alice Aubert, Petra Drnkova, Maxime Chaillet, Isabelle Kolb, Natalie Troffer-Charlier, and Jean-Marie Garnier for sharing their experience on baculovirus expression and molecular biology. This work was funded by the CNRS, the INSERM, the Université de Strasbourg (UdS), the Alsace Region, and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI) and supported by national member subscriptions. It benefited from grants ANR-12-BSV8-0015-01 from the Agence Nationale de la Recherche, INCA-2008-041 from the Institut National du Cancer, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale (FRM) (ING20101221017), and La Ligue contre le Cancer (fellowship to LR). IB acknowledges support from the European Commission (EC) Framework Programme (FP) 7 project ComplexINC (279039).