Protein complex expression by using multigene baculoviral vectors.

摘要: Elucidation of the molecular basis protein-interaction networks, in particular higher eukaryotes, is hampered by insufficient quantities endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment both labor and materials before expression, after biochemical analysis these do not provide flexibility for expressing an altered complex. To meet demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed eukaryotic 1 . Here describe new transfer vectors combination DNA recombination–based methods, which further facilitate generation multigene cassettes protein coexpression (Fig. 1), thus providing flexible platform their rapid regeneration revised studies. Genes encoding components complex are inserted into suite compatible homologous recombination. These progenitor constructs then rapidly joined desired Cre-loxP–mediated vitro plasmid fusion. Protocols integration resulting MultiBac baculoviral genome provided that rely on Tn7 transposition and/or Cre-loxP reaction carried out vivo Escherichia coli cells tailored this purpose. Detailed guidelines virus amplification, cell culture maintenance production provided, together with data illustrating simplicity remarkable robustness present method using composite vector.