Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli.
Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli.
复制标题
在大肠杆菌中生产单跨膜蛋白的新方法的表征。
DOI：
10.1002/bit.26895
复制
发表时间：
2019
影响因子：
3.8
通讯作者：
Smith SM
中科院分区：
文献类型：
--
作者：
Smith SM
重组蛋白的大规模生产和分离是生物技术产业的核心要素，许多产品已被证明对治疗医学非常有益。大肠杆菌是治疗应用中表达外源蛋白的首选微生物，以及一系列高价值蛋白已经通过表征良好的Sec蛋白输出途径靶向外周质。最近，第二种主流的蛋白质输出系统，双精氨酸转位酶，不仅能够将完全折叠的蛋白质转运到ye的外周质中。大肠杆菌，以及其他革兰氏阴性细菌，已经引起了生物技术行业的兴趣。在这项研究中，我们使用了一种新颖的方法来阻止外源Tat底物在输出过程的后期阶段的输出，从而产生一种可溶性结构域位于内膜质周侧的单跨膜蛋白。采用生化和免疫电镜方法研究了双精氨酸转位酶对人类生长激素的输出，以及单跨膜嵌入变异的产生。这是第一次用这种机器输出真正的生物技术相关蛋白质，并以这种方式直接可视化。这里的数据展示了一种生产单跨膜蛋白inE的新方法。杆菌。
The large‐scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine.Escherichia coliis the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high‐value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin‐arginine translocase, to transport fully‐folded proteins into the periplasm of not onlyE. coli, but also other Gram‐negative bacteria, has captured the interest of the biotechnology industry.In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single‐span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno‐electron microscopy approaches were used to investigate the export of human growth hormone by the twin‐arginine translocase, and the generation of a single‐span membrane‐embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single‐span membrane proteins inE. coli.