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Protein complex expression by using multigene baculoviral vectors


Elucidation of the molecular basis of protein-interaction networks, in particular in higher eukaryotes, is hampered by insufficient quantities of endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment in both labor and materials before multiprotein expression, and after expression and biochemical analysis these methods do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression1. Here we describe new transfer vectors and a combination of DNA recombination–based methods, which further facilitate the generation of multigene cassettes for protein coexpression (Fig. 1), thus providing a flexible platform for generation of protein expression vectors and their rapid regeneration for revised expression studies. Genes encoding components of a multiprotein complex are inserted into a suite of compatible transfer vectors by homologous recombination. These progenitor constructs are then rapidly joined in the desired combination by Cre-loxP–mediated in vitro plasmid fusion. Protocols for integration of the resulting multigene expression cassettes into the MultiBac baculoviral genome are provided that rely on Tn7 transposition and/or Cre-loxP reaction carried out in vivo in Escherichia coli cells tailored for this purpose. Detailed guidelines for multigene virus generation and amplification, cell culture maintenance and protein production are provided, together with data illustrating the simplicity and remarkable robustness of the present method for multiprotein expression using a composite MultiBac baculoviral vector.

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Figure 1
Figure 2: Creating multigene transfer vectors.
Figure 3: Stability of MultiBac baculoviral DNA analyzed by ethidium bromide–stained 0.8% agarose gels.
Figure 4: Protein complex expression by using MultiBac baculoviral vector.

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We thank Y. Hunziker for technical assistance, as well as D. Böhringer and C. Ostermeier for helpful comments. D.J.F. was a Human Frontier Science Program fellow, C.S. was a postdoctoral fellow of the Ernst Schering Research Foundation and P.B. was supported by the Swiss National Fund through a grant to U. Suter. T.J.R. acknowledges support from the Swiss National Fund through membership in the National Center of Competence in Research Structural Biology. I.B. acknowledges support from the Swiss National Fund.

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Correspondence to Imre Berger.

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D.J.F., T.J.R. and I.B. are the authors of a US patent describing the MultiBac expression technology (WO2005085456).

Supplementary information

Supplementary Fig. 1

Plasmid maps of MultiBac transfer vectors. (PDF 141 kb)

Supplementary Fig. 2

Plasmid fusions. (PDF 95 kb)

Supplementary Methods (PDF 103 kb)

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Fitzgerald, D., Berger, P., Schaffitzel, C. et al. Protein complex expression by using multigene baculoviral vectors. Nat Methods 3, 1021–1032 (2006).

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