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

背景和意义在实验室和工业中，毕赤酵母越来越多地被用作多种重组蛋白表达的宿主。它能够成功地生成感兴趣的产品，从而简化了整个处理过程。然而，得到的产品滴度通常不是很高。因此，大多数工艺包括高细胞密度发酵，以允许生产大量产品。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，高生物量需要一个稀释步骤，以适应大多数回收步骤，如离心。目前HCD处理面临着几个挑战。细胞活力的延长2。蛋白表达增强影响肉汤中污染物水平（如细胞碎片）和蛋白酶作用的细胞活力是细胞设计和加工条件的结果，即外源蛋白生产施加的压力、培养时间长度、潜在的pH值和细胞对诱导剂的适应性。此外，所需分子的“质量”（例如鲁棒性和物理化学特性）与工艺条件之间的相互关系尚未很好地确定。目的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，减轻下游加工负担，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高级生化工程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