Pichia pastoris protein secretion: analysis of constraints optimisation and methods development

近年来，我们看到进入市场和正在开发的生物制药产品的数量显着增加。它们通常是以蛋白质为基础的，可能由天然蛋白质组成，如胰岛素或治疗剂，它们利用抗体的亲和力将化合物靶向特定组织。这些生物药物都不是很容易从自然来源获得的，所以有必要在细胞中以外来(异源)蛋白的形式生产它们，这样才能大规模生长。除了生产大量蛋白质外，质量稳定(即异质性程度较低)也很重要，以满足监管当局的批准。这不仅可以指蛋白质，也可以指在细胞输出过程中添加到它上面的糖(糖基化)。为了满足这些需求，已经开发了许多细胞类型来表达异源蛋白，包括细菌、酵母、真菌、昆虫和动物细胞。酵母菌特别有用，因为像细菌一样，它们的生长很简单，但它们拥有与动物细胞相同的基本蛋白质输出机制。因此，除非蛋白质有复杂的糖基化模式，这需要在动物细胞中生产，否则在酵母中生产是一个有吸引力的选择。巴斯德毕赤酵母已经发展成为一种非常高效的生产系统，有报道称该系统具有很高的异源蛋白质产量。然而，一些蛋白质不能很好地表达，一个常见的原因是蛋白质被困在出口途径中。这会导致细胞诱导一种名为未折叠蛋白反应(UPR)的应激反应，这实际上会导致粘在系统中的蛋白质降解，从而降低表达水平。(这种反应实际上存在于所有真核细胞中，但当细胞表达外源蛋白时，这种反应更有可能)。虽然普遍定期审议有一些特点，但调查起来相当费力。因此，该项目的主要目标之一是开发一个或多个“报告”系统，该系统指示由于阻塞出口路径而开始诱发普遍定期审议的时间。记者需要具有易于测量的信号，如特定波长的光的吸光度或发射率，或直接化学分析产生的信号的特征模式。一旦我们找到并开发了基于灵敏度和易分析性的最佳报告系统，我们将展示其在实验室规模生产系统中监测和控制UPR的用途，并还将用于筛选多个小规模培养物，以发现哪些诱导或不诱导UPR(以及诱导条件)。此外，使用灵敏和易于测量的报告系统，应该可以筛选其中不诱导UPR的分泌蛋白的变体，并通过这种方式了解导致UPR诱导的蛋白质的特征。即使在普遍定期审议没有问题的情况下，提高蛋白质生产的速度也是有用的。因此，我们还将使用各种技术，其中一些技术最近才对这种有机体可用，以了解是什么限制了分泌良好的蛋白质的分泌。这也可能被证明是普遍定期审议的影响，但也可能是由于系统各部分的能力有限(例如前体的生物合成)，这可以通过营养手段或代谢工程来改善。总体而言，该项目旨在加快工艺开发，从瞄准有用的产品到适合测试和商业化的规模生产。

In recent years we have seen a significant increase in the number of biopharmaceutical products coming on to the market and in development. These are generally protein based and may consist of natural proteins such as insulin or therapeutic agents which exploit the affinity of antibodies to target a compound to a specific tissue. None of these biopharmaceuticals can readily be obtained from natural sources, so it is necessary to produce them as foreign (heterologous) proteins in cells which can be grown on a large scale. As well as producing large amounts of protein it is also important that it is of a consistent quality (ie a low degree of heterogeneity) to meet the approval of regulatory authorities. This may refer not only to the protein but also to the sugars which are added to it (glycosylation) in the process of export from the cell. To meet these needs a number of cell types have been developed for expression of heterologous proteins, including bacteria, yeasts, fungi, insect and animal cells. Yeasts are particularly useful as, like bacteria, their growth is simple, but they have the same basic machinery for protein export as animal cells. So unless a protein has a complex pattern of glycosylation, which would require production in animal cells, production in a yeast is an attractive option. The yeast Pichia pastoris has been developed into a very efficient production system, and there are reports of high heterologous protein yields from this system. However, some proteins do not express well and a common cause is that the protein gets stuck in the export pathway. This causes the cell to induce a stress response called the unfolded protein response (UPR), which can actually lead to degradation of the protein stuck in the system, so reducing the level of expression. (This response is actually found in all eukaryotic cells, but is more likely when the cells are expressing a foreign protein). While there are some characteristic features of the UPR they are quite laborious to investigate. So one of the principle aims of this project is to develop one or more 'reporter' systems, which indicate when the UPR is starting to be induced as a result of blocking the export pathway. Reporters need to have an easily measurable signal, such as absorbance or emission of light at a particular wavelength, or a characteristic pattern of signals arising from direct chemical analysis. Once we have found and developed the best reporter system, based on sensitivity and ease of analysis, we will demonstrate its use for monitoring and controlling the UPR in lab scale production systems and also for screening multiple small scale cultures to discover which induce or do not induce the UPR (and also the conditions under which it is induced). Furthermore, with a sensitive and easily measurable reporter system it should be possible to screen for variants of the secreted protein in which the UPR is not induced and, in this way, understand the features of a protein, which lead to induction of UPR. Even where there is no problem with the UPR, improvement in the rate of protein production would be useful. So we will also use a variety of techniques, some of which have only recently become available for this organism, to understand what might be limiting the secretion of well-secreted proteins. This might turn out also to be the effects of UPR, but may also be due to limitations in the capacity of parts of the system (eg biosynthesis of precursors), which could be improved by nutritional means or through metabolic engineering. Overall, the project aims to speed up process development from the point of targeting a useful product to production on a scale suitable for testing and commercialisation.