Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway

Douglas F Browning; Kirsty L Richards; Amber R Peswani; Jo Roobol; Stephen J W Busby; Colin Robinson

doi:10.1002/bit.26434

Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway

Douglas F Browning, Kirsty L Richards, Amber R Peswani, Jo Roobol, Stephen J W Busby, Colin Robinson

Research output: Contribution to journal › Article › peer-review

Abstract

Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L-1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

Original language	English
Pages (from-to)	2828-2836
Number of pages	9
Journal	Biotechnology and Bioengineering
Volume	114
Issue number	12
Early online date	26 Aug 2017
DOIs	https://doi.org/10.1002/bit.26434
Publication status	Published - Dec 2017

Bibliographical note

© 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Funding: Biotechnology and Biological Sciences Research Council. Grant Number: BB/M018288/1

Keywords

Escherichia coli/genetics
Gene Products, tat/genetics
Genetic Enhancement/methods
Growth Hormone/biosynthesis
Humans
Metabolic Networks and Pathways/genetics
Periplasm/metabolism
Promoter Regions, Genetic/genetics
Recombinant Proteins/biosynthesis
Secretory Pathway/genetics

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10.1002/bit.26434Licence: CC BY 3.0

Escherichia coli TatExpress strains super‐secrete human growth hormone into
© 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Final published version, 1.48 MBLicence: CC BY 3.0

Cite this

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abstract = "Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L-1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.",

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AU - Busby, Stephen J W

AU - Robinson, Colin

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Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway

Abstract

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Keywords

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