Protein complex expression by using multigene baculoviral vectors (original) (raw)

Nature Methods volume 3, pages 1021–1032 (2006)Cite this article

Abstract

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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Acknowledgements

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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  1. Philipp Berger
    Present address: Laboratory of Biomolecular Research, Molecular Cell Biology, Paul Scherrer Institute, CH-5232, Villigen PSI, Switzerland

Authors and Affiliations

  1. ETH Zürich, Institut für Molekularbiologie und Biophysik, ETH-Hönggerberg, CH-8093, Zürich, Switzerland
    Daniel J Fitzgerald, Christiane Schaffitzel, Kazuhiro Yamada, Timothy J Richmond & Imre Berger
  2. ETH Zürich, Institut für Zellbiologie, ETH-Hönggerberg, CH-8093, Zürich, Switzerland
    Philipp Berger

Authors

  1. Daniel J Fitzgerald
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  2. Philipp Berger
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  3. Christiane Schaffitzel
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  4. Kazuhiro Yamada
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  5. Timothy J Richmond
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  6. Imre Berger
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Correspondence toImre 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).

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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). https://doi.org/10.1038/nmeth983

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