College of Health and Life Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK.
Department of Chemistry and Molecular Biology, Gothenburg University, Box 462, 405 30 Göteborg, Sweden.
Biochim Biophys Acta Biomembr. 2021 Sep 1;1863(9):183650. doi: 10.1016/j.bbamem.2021.183650. Epub 2021 May 18.
Aquaporin water channels facilitate the bi-directional flow of water and small, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades, the availability of high-quality protein has underpinned progress in the structural and functional characterization of these water channels. In particular, recombinant protein technology has guaranteed the supply of aquaporin samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression, purification and characterization strategies that have underpinned these successes and that will drive further breakthroughs in the field. Overall, Escherichia coli is a suitable host for prokaryotic isoforms, while Pichia pastoris is the most commonly-used recombinant host for eukaryotic variants. Generally, a two-step purification procedure is suitable after solubilization in glucopyranosides and most structures are determined by X-ray following crystallization.
水通道蛋白促进了所有生命领域中沿着渗透梯度的水和小的、中性溶质的双向流动。在过去的二十年中，高质量蛋白质的可用性为这些水通道的结构和功能特性的研究提供了支持。特别是，重组蛋白技术保证了供应足够质量和数量的水通道蛋白样品，以进行进一步的研究。在这里，我们回顾了成功的表达、纯化和表征策略的特点，这些策略支持了这些成功，并将推动该领域的进一步突破。总的来说，大肠杆菌是原核同工型的合适宿主，而巴斯德毕赤酵母是真核变体最常用的重组宿主。通常，在葡糖苷溶解后采用两步纯化程序，大多数结构通过结晶后的 X 射线确定。
