Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Protein complex expression by using multigene baculoviral vectors

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. 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 

  2. 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 

  3. 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 

  4. Tolia, N.H. & Joshua-Tor, L. Strategies for protein coexpression in Escherichia coli. Nat. Methods 3, 55–64 (2006).

    Article  CAS  Google Scholar 

  5. Uetz, P. et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).

    Article  CAS  Google Scholar 

  6. Giot, L. et al. A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736 (2003).

    Article  CAS  Google Scholar 

  7. 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 

  8. 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 

  9. 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 

  10. Patterson, G., Day, R.N. & Piston, D. Fluorescent protein spectra. J. Cell Sci. 114, 837–838 (2001).

    CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Imre Berger.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth983

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing