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Updated: Jul 10, 2026

The MultiBac Protein Complex Production Platform at the EMBL
Published on: July 11, 2013
The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant
Sven Nottebaum1, Lin Tan, Dominika Trzaska
1Department of Life Sciences, Division of Cell- and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
Abstract:
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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