Most eukaryotic proteins exist as large multicomponent assemblies with many subunits, which act in concert to catalyze specific cellular activities. Many of these molecular machines are only present in low amounts in their native hosts, which impede purification from source material. Unraveling their structure and function at high resolution will often depend on heterologous overproduction. Recombinant expression of multiprotein complexes for structural studies can entail considerable, sometimes inhibitory, investment in both labor and materials, in particular if altering and diversifying of the individual subunits are necessary for successful structure determination. Our laboratory has addressed this challenge by developing technologies that streamline the complex production and diversification process. Here, we review several of these developments for recombinant multiprotein complex production using the MultiBac baculovirus/insect cell expression system which we created. We also addressed parallelization and automation of gene assembly for multiprotein complex expression by developing robotic routines for multigene vector generation. In this contribution, we focus on several improvements of baculovirus expression system performance which we introduced: the modifications of the transfer plasmids, the methods for generation of composite multigene baculoviral DNA, and the simplified and standardized expression procedures which we delineated using our MultiBac system. © 2010 Elsevier Inc.
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