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

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.

SUBMITTER: Nottebaum S

PROVIDER: S-EPMC2248744 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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

Nottebaum Sven S Tan Lin L Trzaska Dominika D Carney Hannah C HC Weinzierl Robert O J RO

Nucleic acids research 20071119 1

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 auto ...[more]

PMID: 18025041

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Dataset Information

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

Publications

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

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