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Automated unrestricted multigene recombineering for multiprotein complex production

Nature Methods volume 6, pages 447–450 (2009)Cite this article

Abstract

Structural and functional studies of many multiprotein complexes depend on recombinant-protein overexpression. Rapid revision of expression experiments and diversification of the complexes are often crucial for success of these projects; therefore, automation is increasingly indispensable. We introduce Acembl, a versatile and automatable system for protein-complex expression in Escherichia coli that uses recombineering to facilitate multigene assembly and diversification. We demonstrated protein-complex expression using Acembl, including production of the complete prokaryotic holotranslocon.

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Figure 1: Multiprotein complex expression with Acembl.
Figure 2: Acceptor-donor recombineering.
Figure 3: Expression of complexes.

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Acknowledgements

We thank R. Jaussi and D. Hart for helpful suggestions, the members of the Berger and Schaffitzel laboratories for discussions and technical assistance, S. Trowitzsch (Max Planck Institute, Göttingen) and the scientists at the Partnership for Structural Biology in Grenoble for providing cDNAs and advice. M.O.S. and T.J.R. are supported by the Swiss National Science Foundation. C.R. and I.B. are supported by the European commission projects Structural Proteomics In Europe 2Complexes (SPINE2C) (European Commission (EC) FP6) and European Infrastructure for Structural Biology INSTRUCT (EC FP7). I.B. is also supported by the Centre National de la Recherche Scientifique (CNRS) and the European commission projects 3D-Repertoire (EC FP6) and Protein Production Platform Pcube (EC-FP7).

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Author notes

  1. Christoph Bieniossek and Yan Nie: These authors contributed equally to this work.

Authors and Affiliations

  1. European Molecular Biology Laboratory, Grenoble Outstation, B.P. 181, Grenoble, France

    Christoph Bieniossek, Yan Nie, Christiane Schaffitzel & Imre Berger

  2. Unit of Virus Host-Cell Interactions, Unités Mixtes de Recherche 5233, Grenoble, France

    Christoph Bieniossek, Yan Nie, Christiane Schaffitzel & Imre Berger

  3. Eidgenössische Technische Hochschule Zürich, Institut für Molekularbiologie und Biophysik, Hönggerberg, Zürich, Switzerland

    Christoph Bieniossek & Timothy J Richmond

  4. Department of Applied Physics, Royal Institute of Technology, Albanova University Center, Stockholm, Sweden

    Yan Nie

  5. Biomolecular Research, Structural Biology, Paul Scherrer Institut, Villigen, Switzerland

    Daniel Frey, Natacha Olieric, Philipp Berger & Michel O Steinmetz

  6. Department of Biochemistry, School of Medical Sciences, Bristol, UK

    Ian Collinson

  7. Department of Biology and Structural Genomics, Institute Génétique et Biologie Moléculaire et Cellulaire, Illkirch, France

    Christophe Romier

Authors
  1. Christoph Bieniossek
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  2. Yan Nie
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  3. Daniel Frey
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  4. Natacha Olieric
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  5. Christiane Schaffitzel
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  6. Ian Collinson
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  7. Christophe Romier
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  8. Philipp Berger
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  9. Timothy J Richmond
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  10. Michel O Steinmetz
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  11. Imre Berger
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Corresponding authors

Correspondence to Michel O Steinmetz or Imre Berger.

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Competing interests

I.B. is inventor on a patent application describing the Acembl technology presented here (European patent application EP 09 154 567.3).

Supplementary information

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Supplementary Figure 1, Supplementary Results, Supplementary Protocol (PDF 3171 kb)

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Bieniossek, C., Nie, Y., Frey, D. et al. Automated unrestricted multigene recombineering for multiprotein complex production. Nat Methods 6, 447–450 (2009). https://doi.org/10.1038/nmeth.1326

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