Development of new-generation bacterial secretion process platforms

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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.