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

近年来，我们已经看到了生物制药产品的数量显着增加，进入市场和开发。这些通常是基于蛋白质的，并且可以由天然蛋白质如胰岛素或利用抗体的亲和力将化合物靶向特定组织的治疗剂组成。这些生物药物中没有一种可以容易地从天然来源获得，因此有必要在可以大规模生长的细胞中将它们作为外源（异源）蛋白质生产。除了生产大量的蛋白质，它具有一致的质量（即低异质性）以满足监管机构的批准也很重要。这不仅可以指蛋白质，而且可以指在从细胞输出的过程中添加到蛋白质中的糖（糖基化）。为了满足这些需要，已经开发了许多细胞类型用于表达异源蛋白，包括细菌、酵母、真菌、昆虫和动物细胞。酵母是特别有用的，因为像细菌一样，它们的生长是简单的，但它们具有与动物细胞相同的蛋白质输出的基本机制。因此，除非蛋白质具有复杂的糖基化模式，这将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有效的生产系统，并且有报道称该系统具有高的异源蛋白产量。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在输出途径中卡住了。这会导致细胞诱导一种称为未折叠蛋白反应（UPR）的应激反应，这实际上会导致系统中蛋白质的降解，从而降低表达水平。(This实际上，在所有真核细胞中都发现了这种反应，但当细胞表达外源蛋白时更有可能）。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，表明何时由于阻断出口途径而开始引发普遍定期审议。报告物需要具有易于测量的信号，例如在特定波长处的光的吸收或发射，或者由直接化学分析产生的信号的特征模式。一旦我们发现并开发了最好的报告系统，基于灵敏度和分析的容易性，我们将证明其用于监测和控制实验室规模生产系统中的UPR，以及用于筛选多种小规模培养物以发现哪些诱导或不诱导UPR（以及诱导UPR的条件）。此外，利用灵敏且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，并且以这种方式了解导致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，提高蛋白质生产率也是有益的。因此，我们还将使用各种技术，其中一些技术最近才用于这种生物体，以了解可能限制分泌良好的蛋白质的分泌的原因。这也可能是普遍定期审议的影响，但也可能是由于系统的部分能力（例如前体的生物合成）的限制，这可以通过营养手段或通过代谢工程来改善。总的来说，该项目旨在加快工艺开发，从针对有用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.