Wiesler Simone C, Weinzierl Robert O J
Department of Life Sciences, Imperial College London.
J Vis Exp. 2012 Aug 26(66):e4110. doi: 10.3791/4110.
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 generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase. Altogether, we generated, purified and analysed 381 mutants--a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results. This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.
X射线晶体学是获取蛋白质结构详细视图的首选方法。此类研究需要通过进一步的生化分析加以补充，以便深入了解结构与功能的关系。寡核苷酸和基因合成技术的进步使大规模诱变策略越来越可行，包括用其他19种氨基酸替换目标残基。功能获得或丧失的表型随后允许得出系统性结论，例如特定残基对催化活性、蛋白质稳定性和/或蛋白质-蛋白质相互作用特异性的贡献。为了将不同的表型归因于突变的性质，而不是波动的实验条件，以可控且可重复的方式纯化和分析蛋白质至关重要。高通量策略以及在机器人液体处理平台上对手动操作流程的自动化，创造了在几乎无需人工干预且错误率极低的情况下执行此类复杂分子生物学程序的机会。在此，我们展示一种以高通量方式纯化His标签重组蛋白的通用方法。在最近的一项研究中，我们将此方法应用于基础转录机制的一个组分TFIIB的详细结构-功能研究。TFIIB在体外对于启动子导向的转录不可或缺，并且对于将RNA聚合酶招募到起始前复合物中至关重要。TFIIB包含一个柔性连接域，该连接域穿透RNA聚合酶的活性位点裂隙。这个连接域赋予TFIIB两种可进行生化定量的活性，即（i）在转录起始的“流产”阶段对催化活性的刺激，以及（ii）对将RNA聚合酶特异性招募到起始前复合物中的额外贡献。我们利用高通量纯化方法在TFIIB连接域内产生单、双和三取代及缺失突变，并随后在功能测定中分析它们对RNA聚合酶催化活性的刺激作用。我们总共产生、纯化并分析了381个突变体——这一任务若手动执行将既耗时又费力。我们对蛋白质进行了多次制备和测定，这使我们能够了解任何实验变化，并让我们清楚地了解结果的可重复性。此方法用作纯化His标签蛋白的通用方案，并且已成功用于纯化其他重组蛋白。它目前针对纯化24种蛋白进行了优化，但可进行调整以纯化多达96种蛋白。
