A new generation of E. coli expression hosts and tools for recombinant protein production

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

The main research challenge addressed in this project is to enhance the UK's capacity for the production of recombinant biologics (biopharmaceuticals) such as antibody fragments, growth factors, hormones and other biologically-based medicines produced from live cells. The market for recombinant biopharmaceuticals is estimated to be over $100 billion p.a. and is predicted to exceed $160 billion by 2015. Sales of antibodies and antibody fragments account for a large proportion of these sales and this is the fastest-growing market in this sector.
Over a third of currently-licensed proteins are produced in E. coli, where 'export' out of the cytoplasm to the periplasm is a favoured strategy. This approach minimises downstream processing (DSP) costs because (i) the target protein can be purified from the relatively simple periplasmic contents, and (ii) this avoids debris and DNA contamination which are serious DSP problems.
E. coli is used because of its genetic malleability, safety record and the ability to rapidly grow large and dense cultures. When E. coli-based systems work well, they can produce 0.5 - 5g protein/litre culture; however, current E. coli production platforms have been largely unchanged for the last decade and are beginning to reach their limits in a number of areas, especially in the production of of biopharmaceuticals that have challenging folding or assembly pathways. Many products either form insoluble inclusion bodies in the cytoplasm (where recoveries can be as low as 10%) or fail to be exported to the periplasm because the standard export method is only capable of exporting proteins in an unfolded state.
In this project we aim to develop improved E. coli production systems that will be capable of producing an unprecedented range of target mocules, while delivering products of very high 'quality' in terms of minimal heterogeneity and high folding integrity. We will achieve this increase in purity by focusing on 3 key areas of upstream production and applying innovative solutions to known problems in each area. Success in each individual section on its own will enhance the quality of DSP feed, while synergies between partners will lead to the development of an integrated platform that incorporates all 3 innovations.

1. Transcriptional control: current E. coli production platforms have been largely unchanged for the last decade relying on a relatively small number of promoters. The latest discoveries in transcriptional control will be incorporated into E. coli to allow much-improved control of biotherapeutic production, reducing problems such as overproduction which leads to mis-folding and aggregation. These new constructs, backed up by state of the art 'omics data, will also provide new routes for producing those products that have proved to be recalcitrant to production in E. coli.
2. Sensing protein folding: the Tat secretion system exports folded proteins and thereby provides a method for secreting a new range of products into the periplasm. We will develop E. coli strains that export a range of biopharmaceuticals with high yield and product quality.
3. Styrene Maleic Acid (SMA) co-polymer provides a more specific and efficient release system for periplasmic proteins, yielding a feed that is low in cytoplasmic contaminants. This method provides a powerful new means of releasing biopharmaceuticals that have been exported to the periplasm.

Throughout the project we will work with industrial collaborators to ensure that the strains are validated and fit for purpose.

IN SUMMARY, we will provide industry with three key innovations, each of which is powerful in its own right. Equally importantly, the consortium will combine these innovations to create a wholly-novel production pipeline with unique capabilities.

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
 
Description This grant started 4 years ago and progress has been good. Together with the University of Birmingham collaborators we have developed and are testing new E. coli strains that produce high-value proteins at a range of levels, and which export them to the periplasm by the Tat pathway at enhanced rates. This means that we can produce high-value proteins in the periplasm using this novel pathway, at levels that are industrially-relevant.
With the University of Sheffield collaborators we have used protemic approaches to analyse the new strains in great detail. These data are being written up for publication.
The novel strains have been distributed to a range of companies and Institutions.
We are just in the process of publishing the key publications from the grant where we show that the strains export 2 biopharmaceuticals at levels of 1-5 grams protein per litre of culture - these are very high levels.
Exploitation Route We are collaborating with a range of biotechnology companies to commercialise these findings, and UCB have hosted team members to test our new strains in fermentation analyses
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Influence on Institutional attitudes to international research collaborations
Geographic Reach Asia 
Policy Influence Type Influenced training of practitioners or researchers
 
Description GCRF establishment of biopharmaceutical and animal vaccine production capacity in Thailand and neighbouring South East Asian countries
Amount £4,900,000 (GBP)
Funding ID BB/P02789X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 12/2021
 
Description Marie Curie Initial Training Network 'ProteinFactory'
Amount £3,500,000 (GBP)
Funding ID 642836 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 05/2015 
End 04/2019
 
Title New E. coli strains 
Description We have developed new strains of E. coli that export proteins to the periplasm by a novel pathway 
Type Of Material Cell line 
Year Produced 2010 
Provided To Others? Yes  
Impact Several groups are testing these strains with a view to using them for production production on analytical or production scale 
 
Description Collaboration with industrial partners 
Organisation UCB Pharma
Department Cellular Sciences - UCB Pharma
Country United Kingdom 
Sector Private 
PI Contribution Team members visited UCB to carry out fermentation analysis
Collaborator Contribution UCB provided state of the art fermentation facilities and staff to help run the experiment bs
Impact Outputs will materialise later this year
Start Year 2017
 
Title Tat Expression System 
Description The present invention describes an expression system for the production of recombinant proteins, in particular a twin arginine translocase Tat system 
IP Reference WO2019038555 
Protection Patent application published
Year Protection Granted 2018
Licensed Commercial In Confidence
Impact Not applicable
 
Description 4th International Biotechnology and Bioengineering Conference, Kuala Lumpur 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was an international conference on industrial biotechnology, attended by a wide range of academics, companies and stakeholders. I was asked to give a Plenary presentation on our research and our GCRF project
Year(s) Of Engagement Activity 2019
URL https://bioscienceconference.com/biotech-2019/
 
Description Open day presentations 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Hosted open day visits to my lab in majority of UCAS days - 2 groups of UCAS applicants + parents per open day, at least 10 open days per year

Increased understanding of biotech projects at Kent
Year(s) Of Engagement Activity 2007,2009,2011,2012,2013
 
Description School visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk sparked questions and discussion

Not applicable
Year(s) Of Engagement Activity 2010,2011,2015