Development of new-generation bacterial secretion process platforms

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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.

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.