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Baculovirus expression system for heterologous multiprotein complexes

Abstract

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.

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Acknowledgements

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). https://doi.org/10.1038/nbt1036

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