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

细菌E.大肠杆菌是用于制备商业上相关的蛋白质例如重组胰岛素的标准宿主。然而，在E.杆菌由于这些蛋白质中的大多数不是来源于大肠杆菌。大肠杆菌中，并产生的水平远远高于E。大肠杆菌通常会产生任何天然蛋白质，E.大肠杆菌通常通过将蛋白质以非活性形式储存在称为包涵体的细胞隔室中来处理它们。蛋白质在包涵体中的积累导致商业生产的问题，包括以活性构象回收蛋白质的昂贵步骤。为了克服这些问题，一些蛋白质在细胞的另一个称为周质的隔室中产生。然而，如果在周质中产生太多的蛋白质，它会使细胞破裂，在此过程中杀死细胞，因此很少或没有蛋白质产生。即使是那些在周质中产生时稳定的，也需要昂贵的纯化，因为有许多其他的天然E。这部分细胞中的大肠杆菌蛋白。当表达非天然蛋白质时，E.大肠杆菌细菌将蛋白质放置在细胞外-这可以防止蛋白质在包涵体或周质中积累，这可能导致细胞死亡。此外，E.大肠杆菌在细胞外不产生或产生很少的天然蛋白质。因此，当蛋白质在细胞外产生时，它们相对干净，几乎不需要清理。然而，由于许多技术原因，已经证明在细菌细胞外生产蛋白质是非常困难的。在这里，我们将利用一个非常简单的系统，称为自动转运系统，该系统具有在细胞外产生非天然蛋白质的良好记录。我们将尝试优化一些商业相关蛋白质的系统。

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.