Development of new-generation bacterial secretion process platforms

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.

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.

This is a synthetic biology project based on novel recent data, which will be of direct benefit to a wide cross section of academic and industrial groups.

A. Impact on industry and society.
The global therapeutic protein market was valued at $93 billion in 2010 and is forecast to reach $140 billion by 2017. Recombinant antibodies are a rapidly growing sector but other proteins are also key reagents for new therapies. Over 30% of licensed therapeutic proteins are produced in E. coli, which is a core platform for this industry. Many E. coli-generated proteins are currently purified after secretion into the periplasm by the 'Sec' protein export pathway, to simplify downstream processing. This application involves new strategies based on the Tat export system that offer major advantages over current Sec-based approaches. Our BRIC1 work has shown for the first time that the Tat system can definitely export proteins at levels required by industry. It is now ready for full exploitation.

Because the market for recombinant proteins is so large, there is an urgent need for new production tools and new, more cost-effective production protocols. Many recombinant proteins contain disulphide bonds and many have been difficult to produce due to expression problems. The first strand of this project will generate new strains that (i) express proteins with disulphide bonds correctly formed in the cytoplasm, and (ii) then export them out of cytoplasm. These strains offer huge potential for industrial applications because the Tat system only exports correctly-folded proteins, leading to high product quality. Preliminary data show that Tat exports S-S bonded proteins very well.

The other main strand of work, exploiting a TatAdCd-based protein secretion platform, offers another unique production system that allows harvesting of diverse protein products directly from the culture medium for the first time. This production method will be useable for a wide range of proteins.

The overall programme will furthermore provide the framework for comparing different production systems and their potential ease of manufacturability with an insight into costs involved. The key impact will be 'right quality product for right use'.

A large cross-section of groups will be able to exploit these techniques, and we predict direct benefits within the biotechnology industry:
(i). Collaborations with BRIC companies are already underway, and additional collaborations will be actively sought.
(ii). We are in discussions with other UK companies, with whom other collaborations will be initiated.

B. Impact on academic research groups
The impact on academic research should also be high. A vast range of research projects require highly-purified protein in moderate (multi-mg) amounts; examples include biophysical studies, protein-protein interaction work, structural studies (especially crystallisation and NMR work). The TatAdCd system produces high levels of protein even in shake flask culture, 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.

C. Net impact of the work
The impact of this research can be summarised as follows:
(i). New reagents can be produced, with high quality.
(ii). More cost-effective production methods for existing or potentially novel biopharmaceuticals.

The actual impact could be very high indeed. The market for proteins that are extracted from the E. coli periplasm after Sec-dependent export is currently worth several $billion per year. If Tat-dependent export systems occupy even 10% of this market, this would mean the Tat system underpinning a market worth several hundred $million. We believe that this is within reach and we have undertaken to assist groups with take-up of this research (see Pathways to Impact) in order to promote the technology as widely, and as rapidly, as possible.