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Baculovirus expression system for heterologous multiprotein complexes


The discovery of large multiprotein complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant multiprotein production and multigene transfer applications.

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Figure 1: Assembly of polycistronic vectors with the MultiBac system.
Figure 2: Expression of multiprotein complexes.
Figure 3: Fluorescent protein monitors of gene expression.


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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  3. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    CAS  Article  Google Scholar 

  4. Forler, D. et al. An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat. Biotechnol. 21, 89–92 (2003).

    CAS  Article  Google Scholar 

  5. Gavin, A.C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002).

    CAS  Article  Google Scholar 

  6. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    CAS  Article  Google Scholar 

  7. Schwikowski, B., Uetz, P. & Fields, S. A network of protein-protein interactions in yeast. Nat. Biotechnol. 18, 1257–1261 (2000).

    CAS  Article  Google Scholar 

  8. von Mering, C. et al. Comparative assessment of large-scale data sets of protein-protein interactions. Nature 417, 399–403 (2002).

    CAS  Article  Google Scholar 

  9. Grigoriev, A. On the number of protein-protein interactions in the yeast proteome. Nucleic Acids Res. 31, 4157–4161 (2003).

    CAS  Article  Google Scholar 

  10. Alberts, B. The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294 (1998).

    CAS  Article  Google Scholar 

  11. Selleck, W. et al. A histone fold TAF octamer within the yeast TFIID transcriptional coactivator. Nat. Struct. Biol. 8, 695–700 (2001).

    CAS  Article  Google Scholar 

  12. de Felipe, P. Polycistronic viral vectors. Curr. Gene Ther. 2, 355–378 (2002).

    CAS  Article  Google Scholar 

  13. Planelles, V. Hybrid lentivirus vectors. Methods Mol. Biol. 229, 273–284 (2003).

    CAS  PubMed  Google Scholar 

  14. O'Reilly, D.R., Miller, L.K. & Luckow, V.A. Baculovirus expression vectors. A laboratory manual. (Oxford University Press, New York, 1994).

  15. Roy, P., Mikhailov, M. & Bishop, D.H. Baculovirus multigene expression vectors and their use for understanding the assembly process of architecturally complex virus particles. Gene 190, 119–129 (1997).

    CAS  Article  Google Scholar 

  16. Bertolotti-Ciarlet, A., Ciarlet, M., Crawford, S.E., Conner, M.E. & Estes, M.K. Immunogenicity and protective efficacy of rotavirus 2/6-virus-like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice. Vaccine 21, 3885–3900 (2003).

    CAS  Article  Google Scholar 

  17. Luckow, V.A., Lee, S.C., Barry, G.F. & Olins, P.O. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli . J. Virol. 67, 4566–4579 (1993).

    CAS  Article  Google Scholar 

  18. Bac-to-Bac Baculoviorus Expression Systems Manual. (Invitrogen, Life Technologies Inc., 2000).

  19. Liu, Q., Li, M.Z., Leibham, D., Cortez, D. & Elledge, S.J. The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8, 1300–1309 (1998).

    CAS  Article  Google Scholar 

  20. Metcalf, W.W., Jiang, W. & Wanner, B.L. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers. Gene 138, 1–7 (1994).

    CAS  Article  Google Scholar 

  21. Slack, J.M., Kuzio, J. & Faulkner, P. Characterization of v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus. J. Gen. Virol. 76, 1091–1098 (1995).

    CAS  Article  Google Scholar 

  22. Suzuki, T. et al. Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene. J. Gen. Virol. 78, 3073–3080 (1997).

    CAS  Article  Google Scholar 

  23. Hom, L.G. & Volkman, L.E. Autographa californica M nucleopolyhedrovirus chiA is required for processing of V-CATH. Virology 277, 178–183 (2000).

    CAS  Article  Google Scholar 

  24. Fitzgerald, D.J. et al. Reaction cycle of the yeast Isw2 chromatin remodeling complex. EMBO J. 23, 3836–3843 (2004).

    CAS  Article  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  26. Kost, T.A. & Condreay, J.P. Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends Biotechnol. 20, 173–180 (2002).

    CAS  Article  Google Scholar 

  27. Huser, A., Rudolph, M. & Hofmann, C. Incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors. Nat. Biotechnol. 19, 451–455 (2001).

    CAS  Article  Google Scholar 

  28. Zhang, Y., Buchholz, F., Muyrers, J.P. & Stewart, A.F. A new logic for DNA engineering using recombination in Escherichia coli . Nat. Genet. 20, 123–128 (1998).

    CAS  Article  Google Scholar 

  29. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    CAS  Article  Google Scholar 

  30. Shizuya, H. et al. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89, 8794–8797 (1992).

    CAS  Article  Google Scholar 

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We thank Christiane Schaffitzel and Ralf Wellinger for discussions, as well as Philipp Berger, Yvonne Hunziker, Martina Mijuskovic, Carl DeLuca and Robert Coleman for assistance. We appreciate helpful comments on baculovirus expression from Robert Noad and Polly Roy. I.B. was a Liebig fellow of the Fonds der Chemischen Industrie (FCI, Germany), D.J.F. was supported by a Human Frontiers Science Program (HSFP) post-doctoral fellowship. We appreciate support from the Swiss National Fund through membership in the National Center for Competence in Research Structural Biology.

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Correspondence to Timothy J Richmond.

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A provisional US patent application no 60/552.072 has been filed.

Supplementary information

Supplementary Fig. 1

Replacement of chiA and v-cath by LoxP. (PDF 136 kb)

Supplementary Note (PDF 6 kb)

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Berger, I., Fitzgerald, D. & Richmond, T. Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 22, 1583–1587 (2004).

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