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Nucleic Acids Research Advance Access originally published online on November 19, 2007
Nucleic Acids Research 2008 36(1):245-252; doi:10.1093/nar/gkm1044
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Nucleic Acids Research, 2008, Vol. 36, No. 1 245-252
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes

Sven Nottebaum, Lin Tan, Dominika Trzaska, Hannah C. Carney and Robert O. J. Weinzierl*

Department of Life Sciences, Division of Cell- and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK

*To whom correspondence should be addressed. Tel: +44 (0)20 7594 5236; Fax: +44 (0)20 7584 2056; Email: r.weinzierl{at}imperial.ac.uk

Received September 19, 2007. Revised October 19, 2007. Accepted November 1, 2007.

The in-depth structure/function analysis of large protein complexes, such as RNA polymerases (RNAPs), requires an experimental platform capable of assembling variants of such enzymes in large numbers in a reproducible manner under defined in vitro conditions. Here we describe a streamlined and integrated protocol for assembling recombinant archaeal RNAPs in a high-throughput 96-well format. All aspects of the procedure including construction of redesigned expression plasmids, development of automated protein extraction/in vitro assembly methods and activity assays were specifically adapted for implementation on robotic platforms. The optimized strategy allows the parallel assembly and activity assay of 96 recombinant RNAPs (including wild-type and mutant variants) with little or no human intervention within 24 h. We demonstrate the high-throughput potential of this system by evaluating the side-chain requirements of a single amino acid position of the RNAP Bridge Helix using saturation mutagenesis.


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