High-level secretion of a recombinant protein to the culture medium with aBacillus subtilistwin-arginine translocation system inEscherichia coli

Albiniak, Anna M. and Matos, Cristina F.R.O. and Branston, Steven D. and Freedman, Robert B. and Keshavarz-Moore, Eli and Robinson, Colin (2013) High-level secretion of a recombinant protein to the culture medium with aBacillus subtilistwin-arginine translocation system inEscherichia coli. FEBS Journal, 280 (16). pp. 3810-3821. ISSN 1742464X. (doi:https://doi.org/10.1111/febs.12376) (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)

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Official URL
http://dx.doi.org/10.1111/febs.12376

Abstract

The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA–GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L−1 culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing.

Item Type: Article
Uncontrolled keywords: Bacillus subtilis TatAdCd; Escherichia coli ;green fluorescent protein (GFP);twin arginine translocation (Tat) signal peptide
Subjects: Q Science > Q Science (General)
Q Science > QR Microbiology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Colin Robinson
Date Deposited: 10 Jul 2013 09:18 UTC
Last Modified: 14 Jul 2014 12:51 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/34571 (The current URI for this page, for reference purposes)
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