title: 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.
authors.affiliation: Department of Life Sciences, Division of Cell- and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
authors.forename: Sven
authors.initials: S
authors.lastname: Nottebaum
authors.forename: Lin
authors.initials: L
authors.lastname: Tan
authors.forename: Dominika
authors.initials: D
authors.lastname: Trzaska
authors.forename: Hannah C
authors.initials: HC
authors.lastname: Carney
authors.forename: Robert O J
authors.initials: RO
authors.lastname: Weinzierl
mesh_terms: D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012371:Robotics
chemical_list: D019843:Archaeal Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases