School of Life Sciences, University of Warwick, Coventry, UK.
School of Biosciences, University of Kent, Canterbury, UK.
Biotechnol Bioeng. 2019 Apr;116(4):722-733. doi: 10.1002/bit.26895. Epub 2019 Jan 19.
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 coli is 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 only E. 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 in E. coli.
大规模生产和分离重组蛋白是生物技术产业的核心要素，其中许多产品已被证明对治疗药物极有帮助。大肠杆菌是用于表达治疗应用的异源蛋白的首选微生物，并且已经使用特征良好的 Sec 蛋白输出途径将一系列高价值蛋白靶向到周质。最近，第二种主流蛋白输出系统——双精氨酸转运体不仅能够将完全折叠的蛋白运输到大肠杆菌，还能运输到其他革兰氏阴性菌的周质，这引起了生物技术产业的兴趣。在这项研究中，我们使用了一种新颖的方法在输出过程的后期阶段阻断异源 Tat 底物的输出，从而生成一种具有可溶性结构域位于内膜周质侧的单跨膜蛋白。我们使用生化和免疫电子显微镜方法研究了双精氨酸转运体对人生长激素的输出，并生成了一种单跨膜嵌入变体。这是第一次通过该机制直接导出真正的生物技术相关蛋白并进行直接可视化。这里呈现的数据展示了一种在大肠杆菌中生产单跨膜蛋白的新方法。
