The Library
High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilistwin-arginine translocation system in Escherichia coli
Tools
Albiniak, A. M., Matos, Cristina F. R. O., Branston, Steven D., Freedman, R. B., Keshavarz-Moore, Eli and Robinson, Colin (2013) High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilistwin-arginine translocation system in Escherichia coli. FEBS Journal, Volume 280 (Issue 16). pp. 3810-3821. doi:10.1111/febs.12376 ISSN 1742-464x.
Research output not available from this repository.
Request-a-Copy directly from author or use local Library Get it For Me service.
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: | Journal Article | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Subjects: | Q Science > QR Microbiology | ||||||||||||
Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||||||
Library of Congress Subject Headings (LCSH): | Bacillus subtilis, Escherichia coli, Biological transport, Green fluorescent protein | ||||||||||||
Journal or Publication Title: | FEBS Journal | ||||||||||||
Publisher: | Blackwell | ||||||||||||
ISSN: | 1742-464x | ||||||||||||
Official Date: | August 2013 | ||||||||||||
Dates: |
|
||||||||||||
Volume: | Volume 280 | ||||||||||||
Number: | Issue 16 | ||||||||||||
Page Range: | pp. 3810-3821 | ||||||||||||
DOI: | 10.1111/febs.12376 | ||||||||||||
Status: | Peer Reviewed | ||||||||||||
Publication Status: | Published | ||||||||||||
Access rights to Published version: | Restricted or Subscription Access | ||||||||||||
Funder: | Marie Curie Initial Training Networks (ITN), Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC) | ||||||||||||
Grant number: | 215524 (ITN) |
Request changes or add full text files to a record
Repository staff actions (login required)
View Item |