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

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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.