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.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

The static structure of biomacromolecules (proteins, DNA, RNA) defines their function but, under physiological conditions, changes in structure (structural dynamics) are equally important in biological mechanisms. This means that, in order to design molecules that bind to large, flexible biomolecules or which influence the conformations that they adopt in solution, we must have access to accurate structural and dynamic information about the target molecule. Current analytical methods fall into two categories, those that provide detailed structures (X-ray crystallography, cryo-EM, protein-observed NMR), but are time consuming to apply (low throughput) and those that report rapidly on intermolecular interactions but provide little structural insight such as ligand-observed NMR, native mass spectrometry or surface plasmon resonance. A step change in our use of structural and dynamic information is offered by two-dimensional infrared (2D-IR) spectroscopy, which uses a sequence of mid-IR laser pulses to excite molecular vibrations and generate a unique 2D 'map' of the 3D structure, structural dynamics and intermolecular interactions of biological molecules. Crucially, modern laser technology has dramatically shortened the amount of time needed to acquire a 2D-IR spectrum, opening up exciting possibilities for 2D-IR to be used as a high-throughput structure-based screening tool or to probe complex and evolving molecular mixtures in real time. Recently, world-leading research led by York has developed 2D-IR measurements of the structure and dynamics of biological molecules in water (H2O) and biofluids. This invention removes the traditional need for replacement of water with 'heavy water' (D2O) before IR measurements, which is both time consuming and expensive. Moreover, this new ability paves the way to label-free molecular analysis of biofluids without sample drying (Chem Sci, 10, 6448-6456, 2019, Editors' Choice) and 2D-IR protein-drug screening experiments in H2O. We believe that rapid structure/dynamics-based 2D-IR analysis of molecules under physiologically relevant conditions will fill an important gap in our analytical capability, transforming biological chemistry research and providing a new tool for healthcare diagnostics. To exploit this enormous potential, we propose to build a globally unique high throughput 2D-IR instrument at York that can measure microlitre volume samples in under a minute. This new capability will: 1) Advance biomedical diagnostics by quantifying the biomolecular content of biofluids for disease diagnosis without labelling, drying or use of antibodies. 2) Enhance next-generation photonic biosensors by enabling structure-based optimisation of sensor-analyte interactions in biofluids. 3) Deliver enabling technology for chemical biology and drug design via real-time mechanistic insight into molecular synthesis and structure-based screening of candidate molecules binding to proteins and nucleic acids without expensive, laborious replacement of H2O with D2O. 4) Measure structural dynamics of biomolecules and ligands in their native solvent for the first time.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Recombinant biotherapeutics are medicines for diagnosis and treatment of disease. Those of interest in this project are proteins that are built (by cells acting as programmed factories) from knowledge of the body's own macromolecules and are redesigned for the treatment of specific diseases. For example, versions of insulin (for treatment of diabetes) have been designed that have given diabetics much greater control of their health. Other natural molecules (antibodies) are being synthesized to treat cancer and autoimmune diseases. The ability to make biotherapeutics has been driven by industry with the potential for life-changing treatments balanced against the commercial costs of production. A new generation of biotherapeutics (with the potential to treat an increased range of diseases with much greater effectiveness than the existing biotherapeutics) are in development. The new generation is being designed by taking bits of natural molecules and building novel molecules (in modular structures) for production in cell factories. This is what is called synthetic biology, the engineering of new genetic materials for valuable functions. Whilst this has incredible promise and potential, the ability to produce novel biotherapeutics is far from optimal (due to the manner in which the cell factories handle such unnatural products). To harvest the full potential of these novel medicines, there is a need to more fully understand the processes that take place in the cell factory and, with knowledge of those processes, to enhance their production. This project aims to address the fundamentals that will determine the effectiveness of production of novel biotherapeutics and provide new systems that allow the production of these potentially powerful new medicines at a yield and quality not currently possible. In this study, we will investigate these problems using biotherapeutic products that exemplify the pipeline of real potential products under development at UCB (a company that is at the forefront of technologies to manufacture novel biotherapeutics). Overall, the project aims to investigate and determine how the cell factory responds to the challenge of production of a series of novel biotherapeutics. We also aim to redesign the molecules themselves, by adding into the molecules the ability to have sugars modify specific parts of the molecule, to drive quality control mechanisms in the cell factory that will allow the better assembly and production of the target molecules. We will also use the latest approaches to modifying the cell factory itself, so termed genome editing approaches, to allow us to unravel the 'roadblocks' in the cell factory that prevent production of the molecules of interest and that also allow us to manipulate the cell factory in an attempt to overcome any observed roadblocks. Thus, we will generate an understanding of why the cell factory does not produce difficult to express molecules, how the additional of sugars onto the molecule may help the cell factory recognise 'badly made' material and correct this, and new cell factory systems for industrial application for the manufacture of these new medicines. In delivering the project we will bring together two world-leading laboratories studying cell factories and their manipulation and the needs of UCB towards production of novel biotherapeutics. The understanding that will arise from the project has wide-scale potential for (i) fundamental understanding of the manner in which cell factories operate and their selective control of production of biotherapeutics of different structure and (ii) for translation of findings into industrial practice (at UCB and wider) for more rapid, more certain and less costly production of new medicines. The approach developed by this project will have widespread value across the entire commercial sector and will have direct relevance to the potential to treat many clinical conditions.