Selective biochemical and synthetic biology approaches for improved delivery of recombinant proteins to the extracellular milieu

The biotechnology industry produces revenues in excess of £500 billion per year. This industry relies on the production of proteins to produce biopharmaceuticals (novel drugs and vaccines), bioproducts for use in food production and in foods (animal health-care biologics, biological plant-growth promoters and pesticides, nutritional supplements, and food additives), bioremediation to clean up the environment (e.g. enzymes to break down oil after oil spillages) and may offer alternatives to environmentally polluting or fossil-fuel-consuming manufacturing processes (e.g. methods to produce hydrogen from bacteria; industrial catalysts). The standard mechanism for making proteins is to produce them in a non-pathogenic bacterium called E. coli. However, there are limits to the types of protein that can be produced in E. coli. As most of these proteins are not derived from E. coli and are produced at levels much greater than E. coli would normally produce any natural protein, the E. coli bacterium often deals with them by storing the proteins in an inactive form in a compartment of the cell called the inclusion body. The accumulation of proteins in inclusion bodies causes problems for commercial production, including expensive steps to recover the proteins in an active conformation. To overcome these problems some proteins are produced in another compartment of the cell called the periplasm. However, if too much protein is made in the periplasm it can burst the cell open, killing the cell in the process, and therefore little or no protein is produced. Even those that are stable when produced in the periplasm will need expensive purification as there are many other native E. coli proteins in this part of the cell. When expressing a non-native protein the best place for the E. coli bacterium to place the protein is outside of the cell - this prevents accumulation of the protein in inclusion bodies or in the periplasm which might lead to cell death. In addition, the laboratory strains of E. coli produce none, or very little, native proteins outside of the cell. Thus, when proteins are produce outside the cell they are relatively clean and need much less effort and investment to bring them to purity. However, for a number of technical reasons it has proved to be very difficult to produce proteins outside the bacterial cell. Here we will utilise a very simple system called the Autotransporter system which has a proven track record for producing non-native proteins outside of the cell. We have already used this system to produce a proteins relevant to industry proving the system works. We will now attempt to make the system produce proteins at levels which are beneficial to industry.

The explosion of genomic data and development of high-throughput robotic screening are providing unprecedented opportunities for the rational design of therapeutic proteins. Many companies have been successful in expressing recombinant proteins from pathogens for vaccine development e.g. the GSK acellular whooping cough vaccine, while others have been successful in using bacterial vectors to express a wide variety of mammalian derived proteins e.g. anti-TNFalpha from UCB-CellTech. In both cases emphasis is on easily expressed and soluble proteins purified from the cytoplasmic or periplasmic compartments. Furthermore, in both cases target protein accumulates in cellular compartments which also contain a plethora of native host proteins thus necessitating the costly recovery and separation of the target protein from host protein. Such separation technologies usually take the form of chromatographic techniques and/or traditional bind-elute technologies. As a result difficult to express and recover 'problem' proteins are often ignored by therapeutic developers, thereby diminishing the numbers of potentially useful targets for tackling disease. We will exploit the characteristics of the Autotransporter secretion mechanism to deliver a variety of different proteins to the extracellular milieu. We will optimise the Autotransporter system for heterologous protein production using standard molecular biology techniques, followed by approaches developed by Yanina Sevastsyanovich, the named research co-Investigator on this grant. We will utilise synthetic biology approaches to build optimised strains for production of recombinant Autotransporter proteins and we will evolve the native autotransporter system to enhance secretion of the target proteins. Development of this system will be of benefit to all members of the BioProcess community, particularly those interested in the recombinant production of therapeutic proteins.

Worldwide markets for biotechnology-derived products are projected to grow to at least $50 billion per year within the next 10 years. Bioprocessing is responsible for translating life-science discoveries into practical products, processes, or systems capable of serving the needs of society. It is critical in moving newly discovered bioproducts such as biopharmaceuticals into the hands of the consuming public. Biopharmaceutical products already account for almost 15% of the total $500 billion pharmaceutical. The number of licensed biopharmaceuticals is forecast to grow at a rate of around 20% per annum. The UK must sustain and extend its expertise in bioprocessing to enable new product development and to retain a lead position against the rapid pace of activity elsewhere. The development of viable, scalable protein purification techniques is critical to being able to produce and recover proteins for therapeutic uses. The work described here is an important fundamental step in the further development and application of E. coli autotransporters for the synthesis and secretion of hitherto difficult to produce biologically active molecules The system(s) developed in this project will help meet the demand for improved bioprocessing and the technology will be directly relevant to the British Healthcare industry so the results will have broad reaching applications in technologically intensive areas of engineering such as the pharmaceutical, and the emerging biotechnology sectors. This will significantly enhance their competitiveness in a worldwide market place. This research will be of benefit for researchers and industrialists in the biotechnology sector not only in the UK but world-wide. Importantly, the rights to such intellectual property would carry considerable prestige and potentially lead to patents and certainly be worthy of dissemination through publication in learned journals in addition to worldwide symposia. Of particular note, the development of the Autotransporter system to effectively release heterolgous proteins into the supernatant is currently undergoing patenting by the applicants and the Health Protection Agency are currently negotiating a strategic collaboration to trial some of the heterolgous constructs as novel vaccines.