| Autor: |
Nottebaum S; Department of Life Sciences, Division of Cell- and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK., Tan L, Trzaska D, Carney HC, Weinzierl RO |
| Jazyk: |
angličtina |
| Zdroj: |
Nucleic acids research [Nucleic Acids Res] 2008 Jan; Vol. 36 (1), pp. 245-52. Date of Electronic Publication: 2007 Nov 19. |
| DOI: |
10.1093/nar/gkm1044 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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