Development of an integrated system for the production and delivery of recombinant biotherapeutics

The bacterium E. coli is a standard host for making commercially relevant proteins e.g. recombinant insulin. 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 little cleaning up. 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 will attempt to optimise the system for a number of commercially relevant proteins.

Gene cloning and expression rarely limit the production of recombinant proteins required for the production of biopharmaceutical products, but substantial bottlenecks arise from protein folding, post-translational modifications and secretion. Against this background, it is too often assumed that product recovery and downstream processing present minor technical problems that current technology can overcome. Recent advances in chromatographic techniques make such an assumption reasonable, but only if the protein can be delivered from the fermenter in a soluble form. The primary aim of this proposal is therefore to design a general strategy for the accumulation and secretion of biopharmaceutical proteins. The critical innovative steps will be to exploit three unique properties of autotransporters: their ability to secrete themselves out of bacteria into the surrounding culture fluid; their ability to co-transport partner proteins, for example, a desired biopharmaceutical protein, by a piggy back mechanism; and finally their ability to release the transported protein from the autotransporter itself. In this application we have three primary aims (1) We will specifically determine the components of the autotransporter system that are essential to achieve extracellular release of target proteins (2) We will optimise the system for maximal protein production using traditional microbial physiology techniques. At this juncture, the two major requirements for downstream processing are a dewatering step; and the separation of the desired product from the major contaminant, the bacterial cell. (3) We will apply knowledge from the first two aims to optimise a vaccine strain of Salmonella for delivery of biopharmaceutical proteins in vivo.