A Novel and Fast Purification Method for Nucleoside Transporters.
A Novel and Fast Purification Method for Nucleoside Transporters.
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一种新颖且快速的核苷转运蛋白纯化方法。
DOI：
10.3389/fmolb.2016.00023
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发表时间：
2016
影响因子：
5
通讯作者：
Goldman A
中科院分区：
文献类型：
--
作者：
Hao Z;Thomsen M;Postis VL;Lesiuk A;Sharples D;Wang Y;Bartlam M;Goldman A
核苷转运蛋白（NTs）在人类中起着重要的生物学作用，了解核苷转运的分子机制需要高分辨率的结构信息。然而，NTs结构分析的主要瓶颈是用于结晶试验的纯的、稳定的和高质量的天然蛋白质的生产。在这里，我们报告了一种新的膜蛋白表达和纯化策略，包括一个高产量的膜蛋白表达载体的构建，和一个新的和快速的NT纯化协议。这种策略的优点是提高了时间效率，从而获得高质量、活性、稳定的膜蛋白，并有效地使用试剂和耗材。本研究为NTs及其他膜蛋白的研究提供了有益的参考。
Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification.