A new generation of E. coli expression hosts and tools for recombinant protein production
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Biosciences
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
Publications
Ruanto P
(2020)
Activation by NarL at the Escherichia coli ogt promoter.
in The Biochemical journal
Pollock NL
(2018)
Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer.
in Biochimica et biophysica acta. Biomembranes
Lee SC
(2016)
A method for detergent-free isolation of membrane proteins in their local lipid environment.
in Nature protocols
Lee SC
(2016)
Encapsulated membrane proteins: A simplified system for molecular simulation.
in Biochimica et biophysica acta
Hothersall J
(2021)
The PAR promoter expression system: Modified lac promoters for controlled recombinant protein production in Escherichia coli.
in New biotechnology
Hothersall J
(2022)
New vectors for urea-inducible recombinant protein production.
in New biotechnology
Hothersall J
(2022)
Inexpensive protein overexpression driven by the NarL transcription activator protein.
in Biotechnology and bioengineering
Hall SCL
(2018)
Influence of Poly(styrene- co-maleic acid) Copolymer Structure on the Properties and Self-Assembly of SMALP Nanodiscs.
in Biomacromolecules
Hall SCL
(2022)
The interaction of styrene maleic acid copolymers with phospholipids in Langmuir monolayers, vesicles and nanodiscs; a structural study.
in Journal of colloid and interface science
Description | We have developed a new process to reduce the cost of drug production. This involves new strains of bacteria that are optimised to produce drugs at a higher level in a form that is easier to purify alongside new methods to extract drugs from bacteria. |
Exploitation Route | A number of the innovations resulting from the work are being tested by the pharmaceutical industry |
Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Description | Development of an improved SMALP toolkit to extract active membrane proteins |
Amount | £563,000 (GBP) |
Funding ID | BB/S008160/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2019 |
End | 02/2022 |
Description | Investigating GPCR:RAMP interactions using nanobodies |
Amount | £404,022 (GBP) |
Funding ID | BB/R016615/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2019 |
End | 05/2023 |
Description | Unshackling Membrane Protein Research : New Amphiphilic Copolymers for Extraction of Stable, Active Membrane Proteins |
Amount | £492,954 (GBP) |
Funding ID | 223728/Z/21/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2022 |
End | 02/2027 |
Title | Periplasmic Release |
Description | The Use of Styrene Maleic Acid Polymers for specific release of materials from E coli periplasm |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | No |
Impact | NA |
Title | SOLUBILISATION OF MEMBRANE PROTEINS |
Description | Development of a new reagent for extraction of membrane proteins in a stabilised form |
IP Reference | EP2452199 |
Protection | Patent granted |
Year Protection Granted | 2012 |
Licensed | Yes |
Impact | Establishment of a newco (Orbiscope BV) in the Netherlands based on selling materials detailed in the patent. Reagents currently being sold globally to academia and the pharmaceutical industry |