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Characterisation of a novel method for the production of single?span membrane proteins in Escherichia coli

Smith, Sarah M., Walker, Kelly L., Jones, Alexander Stephen, Smith, Corinne J., Robinson, Colin (2018) Characterisation of a novel method for the production of single?span membrane proteins in Escherichia coli. Biotechnology and Bioengineering, . ISSN 0006-3592. (doi:10.1002/bit.26895) (KAR id:70906)

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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.

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 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.

Item Type: Article
DOI/Identification number: 10.1002/bit.26895
Uncontrolled keywords: Biotechnology, Protein engineering, Electron microscopy, Membrane proteins
Subjects: Q Science > QR Microbiology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Colin Robinson
Date Deposited: 10 Dec 2018 15:13 UTC
Last Modified: 08 Dec 2019 00:00 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/70906 (The current URI for this page, for reference purposes)
Robinson, Colin: https://orcid.org/0000-0002-1739-032X
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