Schofield, DM;
Templar, A;
Newton, J;
Nesbeth, DN;
(2016)
Promoter engineering to optimise recombinant periplasmic Fab' fragment production in Escherichia coli.
Biotechnology Progress
, 32
(4)
pp. 840-847.
10.1002/btpr.2273.
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Abstract
Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to the improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterised the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity.
| Type: | Article |
|---|---|
| Title: | Promoter engineering to optimise recombinant periplasmic Fab' fragment production in Escherichia coli |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1002/btpr.2273 |
| Publisher version: | http://dx.doi.org/10.1002/btpr.2273 |
| Language: | English |
| Additional information: | © 2016 American Institute of Chemical Engineers. This is the peer reviewed version of the following article:Schofield, D. M., Templar, A., Newton, J. and Nesbeth, D. N. (2016), Promoter engineering to optimize recombinant periplasmic Fab′ fragment production in Escherichia coli. Biotechnol Progress, 32: 840–847. doi:10.1002/btpr.2273, which has been published in final form at 10.1002/btpr.2273. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1478341 |
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