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bioRxiv · 10.1101/308445

Optimisation of a novel method for the production of single-span membrane proteins in Escherichia coli

Smith, S. M.·Walker, K. L.·Smith, C. J.·Robinson, C.
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Abstract

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 characterised 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 other Gram-negative bacteria, has captured the interest of the biotechnology industry.\n\nIn 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 bona-fide biotechnologically-relevant protein has been exported by this machinery and visualised directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.\n\nHighlightsO_LIThe Tat translocase has captured the interest of the biotechnology industry\nC_LIO_LIBiochemical and immuno-EM approaches showed efficient export of hGH by Tat\nC_LIO_LIA novel approach was used to block export of hGH by Tat in E. coli\nC_LIO_LIWe demonstrate a novel method for producing single-span membrane proteins in E. coli\nC_LI

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Smith, S. M., Walker, K. L., Smith, C. J., Robinson, C.. 2018-04-25. Optimisation of a novel method for the production of single-span membrane proteins in Escherichia coli. https://doi.org/10.1101/308445

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