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Automated unrestricted multigene recombineering for multiprotein complex production


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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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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Correspondence to Michel O Steinmetz or Imre Berger.

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

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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).

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