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.

Planned Impact

As described in proposal submitted to TSB

Publications

10 25 50

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Pollock NL (2018) Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer. in Biochimica et biophysica acta. Biomembranes

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Hothersall J (2022) Inexpensive protein overexpression driven by the NarL transcription activator protein. in Biotechnology and bioengineering

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Gulamhussein A (2019) Examining the stability of membrane proteins within SMALPs in European Polymer Journal

 
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 05/2019 
End 05/2022
 
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