Development of new-generation bacterial secretion process platforms

Lead Research Organisation: University College London
Department Name: Biochemical Engineering

Abstract

Many important therapeutic products are proteins - often termed biopharmaceuticals - that have to be produced in a living organism and then purified. Over 30% of the currently licensed therapeutic proteins are made in the bacterium Escherichia coli, which can be quickly grown in large amounts. Some of these proteins are synthesised in the cell interior (cytoplasm) but a favoured strategy is to 'export' the protein product to the periplasmic space between the two cell membranes. The reasons are two-fold. First, the contents of the periplasm can be extracted relatively easily, by selectively rupturing the outer membrane. Secondly, the periplasm is an oxidising environment, and is thus the only place where disulphide bonds form naturally. These bonds are essential structural features of some proteins.

Industrial applications almost always use the bacterial 'secretory' (Sec) pathway to export the protein product to the periplasm. This system transports the protein through the inner membrane in an unfolded state, after which the protein refolds in the periplasm. This often works very well but the system has serious limitations: some proteins fold too quickly for the Sec system to handle, and others may not fold correctly in the periplasm, which lacks the natural 'chaperone' molecules that normally help most proteins to fold in the cytoplasm.

This application aims to exploit a second bacterial protein export pathway, known as the Tat pathway. This can also export foreign proteins, but the major difference is that it transports proteins in a folded state. Importantly, it appears only to transport proteins in a correctly-folded state, and it therefore offers potential for (i) exporting proteins that the Sec pathway cannot handle, and (ii) producing products of particularly high quality, since they should be correctly folded and hance active.

In a previous project, we showed that E. coli strains over-expressing Tat could export a test protein at very high rates - easily sufficient for industrial applications. This project aims to develop two important variants of these strains, each with unique properties. The project will involve collaboration between Warwick and UCL. The partnership is important: the Warwick group are experienced in Tat studies while the UCL partner is able to rigorously test the quality of strains and their readiness for use by industry.

The first part of the project will create strains that can export prefolded proteins that are disulphide-bonded. Disulphide bonds normally only form in the periplasm, but a Finnish group has developed new E. coli strains which express a thiol oxidase that enables efficient disulphide bond formation in the cytoplasm. Recent collaborative studies have shown that three disulphide-bonded test proteins are efficiently exported by Tat if a signal peptide is attached. These strains offer a new means of producing disulphide-bonded proteins in high quantities, with the potential of generating a product of exceptional folding fidelity.

The second part of the project aims to exploit a surprising recent finding by the applicants' groups. The E. coli Tat pathway normally exports proteins to the periplasm, and the outer membrane almost invariably remains intact during fermentation processes. We have replaced the native E. coli Tat system with a Tat system from Bacillus subtilis (TatAdCd; patent application filed) and have shown that the system also exports proteins to the periplasm with high efficiency. However, during fermentation the outer membrane becomes selectively leaky, and releases periplasmic proteins into the extracellular medium ('broth'). The net result is that even in simple batch fermentations, the broth contains high levels of the protein product and this means that the product can be harvested directly from this broth without the need for extraction of the periplasm. This may be a very cost-effective new means of producing therapeutic proteins.

Technical Summary

E. coli is used to produce over 30% of licensed therapeutic proteins, including antibody fragments, insulins and others. Proteins are often exported to the periplasm to facilitate extraction and allow disulphide bond formation, but many proteins cannot be exported to the periplasm by the standard route involving transport of an unfolded protein by the 'Sec' pathway. In addition, periplasmic extraction still poses many problems for downstream processing. This project uses a different strategy, in which proteins are exported by the Tat pathway. Tat also exports heterologous proteins but they are transported in a fully folded form. Our recent work has shown that Tat can export at very high rates, and we are poised to create two platforms with unique abilities.

The first phase involves a collaboration with Prof L Ruddock (Oulu) who has developed E. coli strains that efficiently form S-S bonds in the cytoplasm. Unlike previously published oxidising-cytoplasm strains, these are robust and suitable for industrial application. This project will engineer E. coli strains that form S-S bonds in the cytoplasm, and then export the folded protein using the Tat system. The strains will be able to handle proteins that are 'Sec-incompatible' and the high fidelity nature of the Tat transport system will result in periplasmic product of high overall quality.

The second phase exploits a key recent finding: that expression of a Bacillus subtilis TatAdCd system in an E. coli tat mutant yields a strain that efficiently exports to the periplasm via Tat, but then releases the product into the culture medium. The research will engineer this strain to be fully ready for industrial use in fermenters. The result will be a new production platform where the target can be purified directly from the culture medium.

In each phase, integrated whole processing properties will be defined for the new strains and a detailed cost of goods assessment will prepare them for full industrial use.

Planned Impact

A. Groups benefitting from this research.

This project will be of direct benefit to a wide cross section of academic and industrial groups. Many recombinant proteins contain disulphide bonds and the use of the new CyDisCo strains offers huge potential for maintaining such proteins in a properly folded state and exporting them out of cytoplasm. The other main strand of work, exploiting the TatAdCd-based protein secretion platform, is a unique new platform that will be useable for a wide range of proteins.

(i). The impact on industry could be very significant: the Sec protein export pathway currently underpins a multi-billion dollar industry, and these new Tat-based systems represent potent new protein export strategies. The Tat system can definitely export proteins at levels required by industry and is now ready for full exploitation.

(ii). The impact on academic research should also be high. A vast range of research projects require highly-purified protein in large (multi-mg) amounts; examples include biophysical studies, protein-protein interaction work, structural studies (especially crystallisation and NMR work). The TatAdCd expression system produces high levels of protein even in shake flask systems, and the exported protein can be purified from the culture medium with ease. We will undertake to assist groups to adopt these secretion techniques throughout the project.


B. Net impact of the work

Given the cross-section of groups who will be able to exploit these techniques, we predict direct benefits within the biotechnology industry in the form of:

(i). New reagents identified and produced.

(ii). More cost-effective production procedures for existing biopharmaceuticals.

The actual level of benefit could be very high: the therapeutic protein market is currently worth approximately $100 billion per year, and recombinant antibodies - a major focus in this project - are the fastest growing product sector.

The benefits to academic groups are harder to predict but they could again be significant.

C. Benefits to BRIC companies

Several BRIC companies are partners in this research and they have expressed a keen interest in the use of Tat to produce recombinant proteins. This project is expected to benefit these and other BRIC companies through the availability of new approaches to protein production.
 
Description we have put in place a system which can potentially speed up and ease the manufacturing of proteins for drug development.
the same technology can also be applied in cases where biorefinery is being developed.
Exploitation Route Industrial collaborations
CobraBio: provided protease-deficient E. coli strains and plasmids encoding 3 constructs (all tested for export).
Fujifilm Diosynth: supplied 8 constructs; all tested.
GSK: supplied 3 constructs; all tested.
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Industrial collaborations CobraBio: provided protease-deficient E. coli strains and plasmids encoding 3 constructs (all tested for export). Fujifilm Diosynth: supplied 8 constructs; all tested. GSK: supplied 3 constructs; all tested. UCB-Celltech: supplied 9 constructs and providing fermentation tests. MedImmune: collaborating on TatAdCd system.
First Year Of Impact 2014
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description Strategy for Emerging Biotechnologies
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact Important regarding development of new strategies for emerging technologies
URL http://www.nuffieldbioethics.org
 
Description industry
Amount £100,000 (GBP)
Organisation PT Bio Farma 
Start 03/2018 
End 03/2021
 
Description COLLABORATION WITH INDSUTTRY 
Organisation Fujifilm
Department Fujifilm Diosynth Biotechnologies
Country United States 
Sector Private 
PI Contribution research testing and demonstration of processing for the production of given molecules
Collaborator Contribution provision of industrially relevant molecules
Impact Successful completion of a PhD leading to the graduate obtaining a position in a related industry.
Start Year 2014
 
Description Collaboration with Stedim ( TAP) 
Organisation Sartorius Stedim Biotech
Country France 
Sector Private 
PI Contribution A very novel high throughput equipment for testing was provided we made significant improvement in its development.
Collaborator Contribution provision of the testing equipment and disposables
Impact The outcome is that the remodelled equipment provides a new generation that can be sued for early process development with rigour
Start Year 2016
 
Description Provision of industrially relevant molecules 
Organisation Cobra Biologics
Country United Kingdom 
Sector Private 
PI Contribution Research testing and demonstration of process options
Collaborator Contribution Provision of industrially relevant molecules
Impact application in manufacturing
Start Year 2014