Novel Tat-based systems for recombinant protein production and surface display in bacteria

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The bacterium Escherichia coli is extensively used to produce proteins for therapeutic use. Example products include therapeutic antibodies, insulin and many others, with a total market of around $100 billion per year. A preferred approach is to 'export' the protein of interest to the periplasmic space between the 2 cell membranes, because purification of the protein is often easier and more cost-effective. Traditionally, protein export is achieved by the 'Sec' pathway, which transports the recombinant protein across the inner membrane in an unfolded form. However, some protein products either cannot refold after transport, or fold too quickly before they are transported, and thus cannot be produced. The Tat protein export pathway offers an alternative approach to the Sec pathway because it transports the protein substrate in a pre-folded form, which bypasses difficult steps involved in unfolding the protein before transport, and refolding it afterwards. We have recently shown for the first time that Tat can export very large quantities of protein.The natural E. coli Tat system exports proteins to the periplasm, just like the Sec system, but we have recently created an E. coli strain that expresses a Tat system (termed TatAdCd) from another bacterium, Bacillus subtilis, in place of the native E. coli Tat system. This strain has unique abilities. It efficiently transports the protein product to the periplasm by the Tat pathway, but then releases the product into the culture medium because it has large holes in its outer membrane. The cells are otherwise robust and can be cultured without problems. This project will use this strain for two purposes.First, we will develop the system for the small- and large-scale production of therapeutic proteins. The strain has enormous potential for this purpose because protein products can be purified directly from the culture medium, bypassing the problematic steps of extraction from the periplasmic space and greatly simplifying downstream processing. This part of the project will also involve use of another new technology, involving expression of a thiol oxidase, in order to export correctly folded disulphide-bonded proteins. Many proteins contain disulphide bonds, and the the thiol oxidase enzyme helps the substrate protein to form disulphide bonds before export. This will help the Tat system to export correctly folded 'high-quality' proteins.Secondly, we will develop a novel system for 'surface display' of proteins and isolation of interacting partner proteins. Many therapeutic proteins are directed against specific protein targets, for example those on cancer cells, and a key method for isolating new therapeutic proteins is to express a massive library of different proteins on the bacterial cell surface and then add labeled target protein. Cells expressing a protein that binds to the target are isolated and the protein of interest is cloned for further testing as a therapeutic protein. Current surface display methods have real limitations, including a requirement that the library of proteins is first transported across the cell membrane by the Sec pathway. We propose to develop a new system using the TatAdCd-expressing cells. In this method, the library of proteins will be exported by the Tat pathway and tethered to the outside of the inner membrane. Because the outer membranes of these cells contain large holes, added labeled target protein will be able to bind to the exposed proteins and interacting partners will be readily identified. This technique should result in the identification of important new therapeutic proteins that have been missed using current techniques.