Bioprocessing research - recombinant proteins

重组蛋白生产（RPP）涉及到一种感兴趣的蛋白质在非原生宿主生物体内的异源过表达。在许多情况下，这个宿主是大肠杆菌。将编码目标蛋白的基因克隆到表达载体上，在转录水平上进行某种形式的诱导控制。构建表达细胞在环境控制条件下，在生物反应器中培养到高密度，并直接合成重组蛋白。最重要的目标是最终纯化出高产量的生物活性产品。它已经发展成为一个价值数十亿英镑的产业，负责大规模生产用于生物治疗、商业应用和研究的蛋白质。在大肠杆菌中合成重组蛋白并非没有警告。蛋白质大，多结构域，需要翻译后修饰，或膜结合，可能是有问题的。任何蛋白质的过度表达都可以促进一些不良的细胞反应。在某种程度上，这是由于RPP的目标与大肠杆菌宿主的内在性质之间的利益冲突。在RPP中，产量最大化是至关重要的。为了正常生长，细胞内环境保持在一种稳态状态，任何对这种状态的扰动都会破坏细胞的基本需求。基因水平上的工艺设计，试图将重组蛋白的合成引导到细胞总蛋白的50%以上。根据重组蛋白的亚细胞定位，许多不同的宿主系统很快变得不堪重负。因此，生物活性和最终产量往往不太理想，许多重组蛋白仍然难以生产。该项目将结合微生物学、分子生物学和生物工艺工程，从分子到生物反应器规模优化重组蛋白的合成。

Recombinant protein production (RPP) involves the heterologous overexpression, of a protein of interest, in a non-native host organism. In many instances, this host is E. coli. A gene encoding the protein of interest, is cloned onto an expression vector, with some form of inducible control at the transcriptional level. Construct expressing cells are cultured to high cell density in bioreactors, under environmentally controlled conditions, and directed to synthesise the recombinant protein. The overriding goal, is the eventual purification of a high yield of biologically active product. It has developed into a multi-billion-pound industry, responsible for the large-scale production of proteins used as biotherapeutics, those with commercial application, and in research. Synthesis of recombinant proteins in E. coli is not without caveats. Proteins that are large, multi-domained, require post-translational modification, or are membrane-bound, can be problematic. Overexpression of any protein can promote a number of undesirable cellular responses. In part, this is due to conflicting interests between the goal of RPP and the intrinsic nature of the E. coli host. In RPP, maximising yield is of critical importance. For proper growth, the intracellular environment is maintained in a state of homeostasis, any perturbation to this subverts the basic needs of the cell. Process design at the genetic level, attempts to direct synthesis of the recombinant protein to upwards of 50% of the total cellular protein. Depending on the sub-cellular localisation of the recombinant protein, a number of different host systems quickly become overwhelmed. Consequently, bioactivity and final yield are often less than desirable, and many recombinant proteins are still difficult to produce. This project will utilise a combination of microbiology, molecular biology and bioprocess engineering to optimise synthesis of recombinant proteins from the molecular to bioreactor scale.