Szmitkowska Agnieszka, Pekárová Blanka, Hejátko Jan
Central European Institute of Technology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic.
Methods Mol Biol. 2020;2077:19-36. doi: 10.1007/978-1-4939-9884-5_2.
Determining conditions optimal for host growth, maximal protein yield, and lysis buffer composition is of critical importance for the efficient purification of soluble and well-folded recombinant proteins suitable for functional and/or structural studies. Small-scale optimization of conditions for protein production and stability saves time, labor, and costs. Here we describe a protocol for quick protein production and solubility screen using TissueLyser II system from Qiagen enabling simultaneous processing of 96 protein samples, with application to recombinant proteins encompassing two intracellular domains of ethylene-recognizing sensor histidine kinase ETHYLENE RESPONSE1 (ETR1) from Arabidopsis thaliana. We demonstrate that conditions for expression and cell lysis found in our small-scale screen allow successful large-scale production of pure and functional domains of sensor histidine kinase, providing a strategy potentially transferable to other similar catalytic domains.
确定宿主生长的最佳条件、最大蛋白产量和裂解缓冲液组成对于高效纯化适用于功能和/或结构研究的可溶性且折叠良好的重组蛋白至关重要。小规模优化蛋白质生产和稳定性条件可节省时间、人力和成本。在此，我们描述了一种使用Qiagen公司的TissueLyser II系统进行快速蛋白质生产和溶解度筛选的方案，该系统能够同时处理96个蛋白质样品，并应用于包含拟南芥乙烯识别传感器组氨酸激酶乙烯反应1（ETR1）两个细胞内结构域的重组蛋白。我们证明，在小规模筛选中发现的表达和细胞裂解条件能够成功大规模生产传感器组氨酸激酶的纯功能结构域，提供了一种可能适用于其他类似催化结构域的策略。
