Insect Cells–Baculovirus System for the Production of Difficult to Express Proteins
The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.
Key wordsBaculovirus Insect cells Homologous recombination Protein expression and production Solubility screen
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).
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