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High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli

Matos, Cristina F.R.O., Branston, Steven D., Albiniak, Anna M., Dhanoya, Arjun, Freedman, Robert B., Keshavarz-Moore, Eli, Robinson, Colin (2012) High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli. Biotechnology and Bioengineering, 109 (10). pp. 2533-2542. ISSN 0006-3592. (doi:10.1002/bit.24535) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided. (Contact us about this Publication)
Official URL
http://dx.doi.org/10.1002/bit.24535

Abstract

Numerous high-value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin-arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed-batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial-type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA-GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co-expressed. In both cases, periplasmic GFP peaked at about the 12?h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60?mg periplasmic GFP per liter culture. The cells over-expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48?h induction period. Fed-batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2?mL?h?1, the cultures reached OD600 values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1?g?L?1 culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec-dependent export approaches. Biotechnol. Bioeng. 2012; 109: 2533–2542. © 2012 Wiley Periodicals, Inc.

Item Type: Article
DOI/Identification number: 10.1002/bit.24535
Uncontrolled keywords: Tat pathway; cell engineering; fermentation; E. coli; protein export
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QR Microbiology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Colin Robinson
Date Deposited: 10 Jul 2013 09:28 UTC
Last Modified: 29 May 2019 10:22 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/34575 (The current URI for this page, for reference purposes)
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