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

大肠杆菌是制造商业相关蛋白质的标准宿主，例如重组胰岛素。然而，在大肠杆菌中可以生产的蛋白质类型是有限制的。由于这些蛋白质中的大多数不是来自大肠杆菌，其产量远远高于大肠杆菌通常产生的任何天然蛋白质的水平，因此，大肠杆菌细菌通常通过将蛋白质以非活性形式存储在细胞的一个称为包涵体的隔间中来处理它们。包涵体中蛋白质的积累给商业生产带来了问题，包括以活性构象恢复蛋白质的昂贵步骤。为了克服这些问题，一些蛋白质是在细胞的另一个称为周质的隔室中产生的。然而，如果周质中产生了太多的蛋白质，它可能会使细胞破裂，在这个过程中杀死细胞，因此产生的蛋白质很少或根本没有。即使是那些在周质中产生时稳定的蛋白质也需要昂贵的纯化，因为在细胞的这一部分有许多其他天然的大肠杆菌蛋白质。当表达非天然蛋白质时，大肠杆菌将蛋白质放置在细胞外的最佳位置--这防止了蛋白质在包涵体或周质中积聚，后者可能导致细胞死亡。此外，实验室的大肠杆菌菌株在细胞外不产生或产生非常少的天然蛋白质。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，由于一些技术原因，在细菌细胞外生产蛋白质被证明是非常困难的。在这里，我们将使用一种非常简单的系统，称为自动转运蛋白系统，它在细胞外生产非天然蛋白质方面有着良好的记录。我们将尝试针对一些商业上相关的蛋白质来优化系统。

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.