Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts
Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts
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DOI：
10.1002/prot.21786
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发表时间：
2008-05-01
影响因子：
2.9
通讯作者：
Lesley, Scott A.
中科院分区：
文献类型：
--
作者：
Klock, Heath E.;Koesema, Eric J.;Lesley, Scott A.
成功的蛋白质表达、纯化和结晶用于挑战性靶标通常需要评估大量表达构建体。通常需要截短和点突变的多次迭代来鉴定用于重组表达的合适衍生物。制造和表征这些变体是成功的重大障碍。我们开发了一种快速有效的克隆方法，并将其与蛋白质微筛选方法相结合，以表征蛋白质结构研究的适用性。使用聚合酶不完全引物延伸（PIPE）克隆方法快速克隆448个蛋白质靶标，然后以最小的努力从96个靶标产生2143个截短。通过微筛选方案表达、纯化蛋白质并进行表征，所述方案结合蛋白质定量、液相色谱质谱和分析性尺寸排阻色谱（AnSEC）以评价蛋白质产物用于X射线晶体学的适用性。结果表明，主要基于表达溶解度选择用于晶体试验的表达构建体是不够的。相反，发现AnSEC评分作为蛋白质多分散性的量度是最终结构确定成功的预测，并且对于确定截断系列的适当边界是必不可少的。通过将这种组合的PIPE克隆和微筛选方法应用于柠檬酸目标，整体结构确定成功率提高了至少38%。
Successful protein expression, purification, and crystallization for challenging targets typically requires evaluation of a multitude of expression constructs. Often many iterations of truncations and point mutations are required to identify a suitable derivative for recombinant expression. Making and characterizing these variants is a significant barrier to success. We have developed a rapid and efficient cloning process and combined it with a protein microscreening approach to characterize protein suitability for structural studies. The Polymerase Incomplete Primer Extension (PIPE) cloning method was used to rapidly clone 448 protein targets and then to generate 2143 truncations from 96 targets with minimal effort. Proteins were expressed, purified, and characterized via a microscreening protocol, which incorporates protein quantification, liquid chromatography mass spectrometry and analytical size exclusion chromatography (AnSEC) to evaluate suitability of the protein products for X-ray crystallography. The results suggest that selecting expression constructs for crystal trials based primarily on expression solubility is insufficient. Instead, AnSEC scoring as a measure of protein polydispersity was found to be predictive of ultimate structure determination success and essential for identifying appropriate boundaries for truncation series. Overall structure determination success was increased by at least 38% by applying this combined PIPE cloning and microscreening approach to recalcitrant targets.