BRIC DOCTORATE PROGRAMME-An upstream platform for the production of high grade heterologous proteins in Pichia pastoris

背景和意义巴斯德毕赤酵母作为宿主在实验室和工业中表达各种重组蛋白的应用越来越多。它能够成功地分泌感兴趣的产物，从而简化整体处理。然而，获得的产物滴度通常不是很高。因此，大多数方法包括高细胞密度发酵以允许制备大量的产物。另一方面，已知高细胞密度会导致细胞活力降低、细胞碎片增加以及无定形和胶体有机成分浓度增加，从而对纯化阶段提出挑战。此外，高生物量需要稀释步骤以适应大多数回收步骤，例如离心。HCD加工目前面临若干挑战（其中包括）1。细胞活力的延长2.蛋白质表达增强3.提高/维持产品质量影响肉汤中污染物（例如细胞碎片）水平和蛋白酶作用的细胞活力是细胞设计和加工条件（即外源蛋白生产、培养物长度以及潜在的pH水平和细胞对诱导物的适应性）的结果。此外，所需分子的“质量”（例如耐用性和理化特性）与工艺条件之间的相互关系尚未得到很好的确立。a）研究培养条件、细胞活力分布和产物表达之间的相互作用，以提高产量，减少下游加工的负担并实现最佳细胞分离。B）调查上游条件对产品质量的影响。在适当的情况下，该项目将利用伦敦大学学院生物化学工程高级中心现有的技术工具箱

Porject title- An upstream platfrom for the production of high grade heterologous proteins in Pichia pastoris Background and significance Pichia pastoris is finding increasing use as a host for the expression of a wide variety of recombinant proteins in the laboratory and in industry. It is capable of successfully secreting the product of interest thus easing overall processing. However, the product titres obtained are usually not very high. As a result most processes include high cell density fermentations to allow large quantities of the product to be made. On the other hand, high cell densities are known to result in reduced cell viability, increased cell debris, as well as an increase in the concentration of amorphous and colloidal organic constituents, thus posing a challenge to the purification stage. In addition the high biomass requires a dilution step to accommodate most recovery steps such as centrifugation. Challenges There are several challenges (amongst others) currently facing HCD processing 1. Extension of cell viability 2. Enhancement of protein expression 3. Enhancement / maintenance of product quality Cell viability which impacts on the level of contaminants in broth (e.g.cell debris) and protease action is the result of both cell design and processing conditions namely stress exerted by foreign protein production, length of culture, and potentially, pH levels and adaptability of cell to the inducer. Furthermore, the interrelation between the 'quality' (e.g. robustness and physico-chemical characteristics) of the desired molecule and the process conditions is not yet well established. Objectives a) To investigate the interaction between culture conditions, cell viability distribution and product expression with the view to enhance production ,reduce the burden on downstream processing and achieve optimal cell separation. b)To investigate the impact of the upstream conditions on product quality. Where appropriate the project will draw on existing tool box of technology available in the UCL Advanced Centre for Biochemical Engineering