A new generation of E. coli expression hosts and tools for recombinant protein production
Lead Research Organisation:
University of Sheffield
Department Name: Chemical & Biological Engineering
Abstract
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Technical Summary
Many biopharmaceuticals are produced in E. coli but current platforms have a number of limitations and cannot produce many potential target products. This project will develop four entirely novel innovations to produce and harvest a wide range of target proteins, delivering new tools and processes that encompass the entire upstream pipeline:
1: Protein synthesis will be driven by a novel set of promoters and inducers that have clear advantages (better inducible control, higher mRNA yield) over currently-used systems. Many of the currently-used inducible promoters for recombinant protein production (RPP) are extremely strong, inherently leaky and present on high-copy number plasmids. RPP often outstrips the ability of the cell to cope, resulting in insoluble aggregates and inclusion body formation.
2: Export to the periplasm will be mediated by an alternative protein export pathway, known as the Tat pathway, that has unique capabilities and clear advantages over the currently-used Sec pathway. A major problem with the Sec pathway is that it transports its substrates in an unfolded state, and cannot handle proteins that fold too quickly or tightly - a significant proportion of potential target molecules. The Tat pathway will instead be exploited to export a wide range of new biotherapeutics in a prefolded form.
3: We will develop and validate a novel method for releasing periplasmic contents which relies on nano-encapsulation of lipids. The method uses a low cost polymer (SMA) which provides a more specific release method than current osmotic shock methods under a wider range of operating conditions.
4: The above innovations will be combined to deliver an integrated platform that is better than the sum of its parts.
The project will be carried out in collaboration with a range of UK companies who will fully validate the new strains and processes.
1: Protein synthesis will be driven by a novel set of promoters and inducers that have clear advantages (better inducible control, higher mRNA yield) over currently-used systems. Many of the currently-used inducible promoters for recombinant protein production (RPP) are extremely strong, inherently leaky and present on high-copy number plasmids. RPP often outstrips the ability of the cell to cope, resulting in insoluble aggregates and inclusion body formation.
2: Export to the periplasm will be mediated by an alternative protein export pathway, known as the Tat pathway, that has unique capabilities and clear advantages over the currently-used Sec pathway. A major problem with the Sec pathway is that it transports its substrates in an unfolded state, and cannot handle proteins that fold too quickly or tightly - a significant proportion of potential target molecules. The Tat pathway will instead be exploited to export a wide range of new biotherapeutics in a prefolded form.
3: We will develop and validate a novel method for releasing periplasmic contents which relies on nano-encapsulation of lipids. The method uses a low cost polymer (SMA) which provides a more specific release method than current osmotic shock methods under a wider range of operating conditions.
4: The above innovations will be combined to deliver an integrated platform that is better than the sum of its parts.
The project will be carried out in collaboration with a range of UK companies who will fully validate the new strains and processes.
Planned Impact
As described in proposal submitted to TSB
People |
ORCID iD |
| Phillip Craig Wright (Principal Investigator) |
Publications
Benninghoff JC
(2021)
Exposure to 1-Butanol Exemplifies the Response of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius to Solvent Stress.
in Applied and environmental microbiology
Bischof LF
(2018)
Early Response of Sulfolobus acidocaldarius to Nutrient Limitation.
in Frontiers in microbiology
Hoffmann L
(2019)
Structure and interactions of the archaeal motility repression module ArnA-ArnB that modulates archaellum gene expression in Sulfolobus acidocaldarius.
in The Journal of biological chemistry
Landels A
(2015)
Advances in proteomics for production strain analysis.
in Current opinion in biotechnology
Stark H
(2017)
Oxidative Stickland reactions in an obligate aerobic organism - amino acid catabolism in the Crenarchaeon Sulfolobus solfataricus.
in The FEBS journal
| Description | Produced on an industrial scale, biotherapeutic proteins including cytokines, growth factors, and hormones, are of high importance and provide crucial therapeutic treatments. To understand how to better optimise the E. coli for this purpose, we here have applied quantitative proteomics tools iTRAQ and LFQ approaches to examine the differently expressed proteomes of two different mutated E. coli strains with targeted proteins (CyDisco proteins and Human Growth Hormone) grown under the presence and absence of IPTG (Isopropyl ß-D-1-thiogalactopyranoside). Two quantitative proteomics approaches have been used to address the question: How could CyDisco proteins regulate E. coli proteomes and recombinant protein. We were able to measure over 1300 proteins and to map these onto metabolic and regulatory pathways. The observations in this investigation will provide useful information for understating the effect of CyDisco in order to improve recombinant protein production via cellular engineering. Papers are currently being written up. |
| Exploitation Route | Currently writing papers for submission. Talking with the biopharmaceuticals industry on findings. |
| Sectors | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Project meetings |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Industry/Business |
| Results and Impact | Engaging with academic and industry representatives on the outcomes of proteome analysis of E.coli cells producing biopharma products. Working out cellular bottlenecks and plans to limit these issues. Likely employment on more industrially relevant examples. |
| Year(s) Of Engagement Activity | 2015,2016,2017 |