Focal Area Structural Biology and Biophysics, Biozentrum, University of Basel, 4056 Basel, Switzerland.
GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 24 Uppsala, Sweden.
J Chromatogr A. 2020 May 10;1618:460846. doi: 10.1016/j.chroma.2019.460846. Epub 2020 Jan 2.
Protein purification processes in basic research using ÄKTA™ liquid chromatography systems are often limited to single sample injections and simple one-column purifications. Because many target proteins in structural biology require complex purification protocols the work easily becomes laborious. To streamline and accelerate downstream protein production, an ALIAS™ autosampler and a modular sample in-line dilution process coupled to ion-exchange chromatography were incorporated into the workflow to automate two of the most commonly performed purification strategies - ion-exchange to size exclusion and nickel-ion metal affinity to size exclusion. The chromatographic setup enabled purification of a large array of cytosolic and membrane proteins from small-scale expression cultures produced in insect cells necessary to develop and optimize isotope-labeling strategies for nuclear magnetic resonance spectroscopy applications, resulting in a reduction in experiment time of about 20% per run for both cytosolic and membrane protein purification schemes. However, when queuing multiple samples the throughput increased by 66% and 75%, respectively. In addition, a novel system configuration is presented, where two column valves can be operated independently. This allows for the design of purification loops to increase purity of the target protein.
在基础研究中使用 ÄKTA™ 液相色谱系统进行蛋白质纯化过程通常仅限于单次进样和简单的单柱纯化。由于结构生物学中的许多靶蛋白需要复杂的纯化方案，因此这项工作很容易变得繁琐。为了简化和加速下游蛋白质生产，将 ALIAS™ 自动进样器和模块化在线样品稀释过程与离子交换色谱结合到工作流程中，以自动化两种最常见的纯化策略 - 离子交换到分子筛和镍离子金属亲和到分子筛。这种色谱设置可从昆虫细胞中小规模表达培养物中纯化大量胞质和膜蛋白，这对于开发和优化用于核磁共振波谱应用的同位素标记策略是必要的，从而使胞质和膜蛋白纯化方案的每个运行实验时间减少约 20%。然而，当排队处理多个样本时，通量分别增加了 66%和 75%。此外，还提出了一种新颖的系统配置，其中两个柱阀可以独立操作。这允许设计纯化循环以提高目标蛋白的纯度。
