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@article{Wiesler:2012:10.3791/4110,
author = {Wiesler, SC and Weinzierl, RO},
doi = {10.3791/4110},
journal = {J Vis Exp},
title = {High-throughput purification of affinity-tagged recombinant proteins},
url = {http://dx.doi.org/10.3791/4110},
year = {2012}
}

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TY  - JOUR
AB  - X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity. In order to attribute the different phenotypes to the nature of the mutation--rather than to fluctuating experimental conditions--it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates. Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex. We exploited the high-throughput purification method to genera
AU  - Wiesler,SC
AU  - Weinzierl,RO
DO  - 10.3791/4110
PY  - 2012///
SN  - 1940-087X
TI  - High-throughput purification of affinity-tagged recombinant proteins
T2  - J Vis Exp
UR  - http://dx.doi.org/10.3791/4110
UR  - http://www.ncbi.nlm.nih.gov/pubmed/22952005
ER  -

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