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 i

SUBMITTER: Nottebaum S 

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

REPOSITORIES: biostudies-literature