Protein complex expression by using multigene baculoviral vectors (original) (raw)
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- Published: 20 November 2006
- Philipp Berger2 nAff3,
- Christiane Schaffitzel1,
- Kazuhiro Yamada1,
- Timothy J Richmond1 &
- …
- Imre Berger1
Nature Methods volume 3, pages 1021–1032 (2006)Cite this article
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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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References
- Berger, I., Fitzgerald, D.J. & Richmond, T.J. Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 22, 1583–1587 (2004).
Article CAS Google Scholar - Benoit, R.M., Wilhelm, R.N., Scherer-Becker, D. & Ostermeier, C. An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids. Protein Expr. Pur. 45, 66–71 (2006).
Article CAS Google Scholar - Tan, S., Kern, R.C. & Selleck, W. The pST44 polycistronic expression system for producing protein complexes in E.coli. Protein Expr. Pur. 40, 385–395 (2005).
Article CAS Google Scholar - Tolia, N.H. & Joshua-Tor, L. Strategies for protein coexpression in Escherichia coli. Nat. Methods 3, 55–64 (2006).
Article CAS Google Scholar - Uetz, P. et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).
Article CAS Google Scholar - Giot, L. et al. A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736 (2003).
Article CAS Google Scholar - Simon, O., Williams, T., Caballero, P. & Lopez-Ferber, M. Dynamics of deletion genotypes in an experimental insect virus population. Proc. R. Soc. Lond. B 273, 783–790 (2006).
Article Google Scholar - Pijlman, G.B., de Vrij, J., van den End, F.J., Vlak, J.M. & Martens, D.E. Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect-cell bioreactors. Biotechnol. Bioeng. 87, 743–753 (2004).
Article CAS Google Scholar - O'Reilly, D.R., Miller, L.K. & Luckov, V.A. eds. Baculovirus expression vectors. A laboratory manual. (Oxford University Press, New York, 1994).
Google Scholar - Patterson, G., Day, R.N. & Piston, D. Fluorescent protein spectra. J. Cell Sci. 114, 837–838 (2001).
CAS Google Scholar
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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- Philipp Berger
Present address: Laboratory of Biomolecular Research, Molecular Cell Biology, Paul Scherrer Institute, CH-5232, Villigen PSI, Switzerland
Authors and Affiliations
- 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 - ETH Zürich, Institut für Zellbiologie, ETH-Hönggerberg, CH-8093, Zürich, Switzerland
Philipp 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
- Published: 20 November 2006
- Issue Date: December 2006
- DOI: https://doi.org/10.1038/nmeth983