Rapid, robotic, small-scale protein production for NMR screening and structure determination.
Rapid, robotic, small-scale protein production for NMR screening and structure determination.
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用于 NMR 筛选和结构测定的快速、机器人、小规模蛋白质生产。
DOI：
10.1002/pro.335
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发表时间：
2010
期刊：
影响因子：
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通讯作者：
Volkman,BrianF
中科院分区：
文献类型：
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作者：
Jensen,DavinR;Woytovich,Christopher;Li,Margie;Duvnjak,Petar;Cassidy,MichaelS;Frederick,RonnieO;Bergeman,LaiF;Peterson,FrancisC;Volkman,BrianF
三维蛋白质结构测定是一个昂贵的过程，部分原因是在潜在靶标组内的成功率较低。传统的验证方法通过耗时的表达、溶解、纯化和折叠筛选，消除了绝大多数蛋白质的进一步考虑。每个阶段的假阴性都会导致总体成功率的无理降低。我们开发了一种半自动化的方案，用于同位素标记蛋白质的生产，使用Maxwell - 16，一种市售的台式机器人，可以通过2D NMR进行单步目标筛选。在一周的时间内，一个人可以表达、纯化和筛选48种不同的15n标记蛋白，将验证过程加快了10倍以上。麦克斯韦- 16单通道的产率足以使用3‐mm样品池和5‐mm低温核磁共振探针获得高质量的2D1H‐15N‐HSQC光谱。麦克斯韦- 16筛选的对照组蛋白质再现了先前的验证结果，这些结果来自传统的小规模表达筛选和我们结构基因组学管道目前采用的大规模生产方法。对18个新蛋白构建体的分析确定了两个潜在的结构靶点，包括人类Par‐3的第二个PDZ结构域。为了进一步证明该生产策略的广泛实用性，我们使用使用Maxwell‐16制备的[U‐15N，13C]蛋白解决了PDZ2 NMR结构。这种新型的半自动化蛋白质生产方案通过消除不必要的筛选和扩大步骤，减少了与核磁共振结构测定相关的时间和成本。
Three‐dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time‐consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi‐automated protocol for isotopically‐labeled protein production using the Maxwell‐16, a commercially available bench top robot, that allows for single‐step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different15N‐labeled proteins, accelerating the validation process by more than 10‐fold. The yield from a single channel of the Maxwell‐16 is sufficient for acquisition of a high‐quality 2D1H‐15N‐HSQC spectrum using a 3‐mm sample cell and 5‐mm cryogenic NMR probe. Maxwell‐16 screening of a control group of proteins reproduced previous validation results from conventional small‐scale expression screening and large‐scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par‐3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U‐15N,13C] protein prepared using the Maxwell‐16. This novel semi‐automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale‐up steps.