Radli Martina, Veprintsev Dmitry B, Rüdiger Stefan G D
Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, CH Utrecht, The Netherlands.
Science for Life, Utrecht University, CH Utrecht, The Netherlands.
PLoS One. 2017 Jun 26;12(6):e0180047. doi: 10.1371/journal.pone.0180047. eCollection 2017.
The molecular chaperone Hsp90 is an essential member of the cellular proteostasis system. It plays an important role in the stabilisation and activation of a large number of client proteins and is involved in fatal disease processes, e.g. Alzheimer disease, cancer and cystic fibrosis. This makes Hsp90 a crucial protein to study. Mechanistic studies require large amounts of protein but the production and purification of recombinant human Hsp90 in Escherichia coli is challenging and laborious. Here we identified conditions that influence Hsp90 production, and optimised a fast and efficient purification protocol. We found that the nutrient value of the culturing medium and the length of induction had significant effect on Hsp90 production in Escherichia coli. Our fast, single-day purification protocol resulted in a stable, well-folded and pure sample that was resistant to degradation in a reproducible manner. We anticipate that our results provide a useful tool to produce higher amount of pure, well-folded and stable recombinant human Hsp90β in Escherichia coli in an efficient way.
分子伴侣Hsp90是细胞蛋白质稳态系统的重要成员。它在大量客户蛋白的稳定和激活中发挥重要作用，并参与致命疾病过程，如阿尔茨海默病、癌症和囊性纤维化。这使得Hsp90成为一个关键的研究蛋白。机制研究需要大量蛋白质，但在大肠杆菌中生产和纯化重组人Hsp90具有挑战性且费力。在此，我们确定了影响Hsp90生产的条件，并优化了一种快速高效的纯化方案。我们发现培养基的营养价值和诱导时间对大肠杆菌中Hsp90的生产有显著影响。我们快速的单日纯化方案产生了一个稳定、折叠良好且纯净的样品，该样品能够以可重复的方式抵抗降解。我们预计我们的结果为在大肠杆菌中高效生产更高量的纯净、折叠良好且稳定的重组人Hsp90β提供了一个有用的工具。
