Understanding and improving the specific productivity of pichia pastoris recombinant protein secretion

了解和提高毕赤酵母重组蛋白分泌的比生产力

• 批准号: BB/F018045/1
• 金额: $ 10.26万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF018045%2F1
• 关键词: Understanding; improving; specific; productivity; pichia

The specific productivity of the majority of recombinant proteins secreted by Pichia pastoris is relatively poor. Identification of high producing recombinant strains is largely an empirical process with little understanding of the characteristics that lead to high productivity on scale up of culture conditions from shake flask scale to high cell density industrial fermentations. Current practice when expressing proteins using Pichia pastoris is to produce recombinants using integrating vectors leading to position independant expression. The most widely used system is based on the alcohol oxidase promoter (AOX) which requires derepression and then induction by methanol. Copy number of the target gene is an important variable but does not necessarily correlate with titre. Selection of the 'best' clone for scale up and manufacturing is complicated by the fact that clones achieving high secreted protein titre in shake flasks do not always achieve high titre in high cell density fermentations. This is also significantly influenced by the impact of methanol utilisation phenotype (mut+, mutS or mut-) of the host. Studies (limited to laboratory processes) have shown that poor expression in Pichia pastoris can in some cases be correlated with folding problems in the ER, which induces an unfolded protein response (UPR). BRIC project 06/18 (David Leak, Imperial College (IC)) is (1) developing metabolomic methods and applying these with transcriptomics to understand the underlying physiology of UPR stressed, non-stressed and non-expressing cultures and, (2) developing easily monitored reporters of UPR to allow ideal expression conditions to be determined on a small scale. This project will use industrially relevant proteins (induced with methanol and non-methanol) and protein models (low and high secretion titres) with an industrial fermentation platform to characterise and better understand the inter-play between the parameters described above and induction of the UPR (UPR tools developed by BRIC 06/18). Metabolomic information (based on extracellular (footprint) metabolites) will also be collected. The student will spend a significant amount of time working at Avecia: 2-3 model proteins, AOX recombinants in range of mut hosts, establishing copy number and titre in shake flask culture, establishing research cell banks of clones for further work (matrix of mut/copy number/titre), establishing fermentation performance of 4-6 clones in 15L fed-batch fermentations - including analysis for footprint metabolites and/or UPR linked GFP expression. The above work is envisaged to take 15 mo with the student spending an equal amount of time at IC (see below) and Avecia. The student will initially work at IC (ca.4 mo) becoming familiarised with the UPR reporter systems being developed and will making constructs (e.g. GFP in various mut backgrounds) necessary for integration into the work at Avecia. The first half (15-18 mo) will define a pattern/decision matrix. Depending on the outcome of this we will either: (1) Expand work with GFP, which provides us with a ready made system for the sorting of variants, making it ammenable for high-throughput analysis to get a more fundamental understanding of the causes of UPR. This coupled with the transcriptomic analysis being done at IC (BRIC 06/18) should point to some host features that can be developed further to improve secretion and protein titre. Or, (2) Investigate the possibility of online control of UPR, which would require taking one/more of the poorly expressing constructs and dissecting the expression conditions to establish the conditions at which specific productivity plateaus/declines and then then investigating whether we can develop a feedback control regime which keeps at this level. The second phase of the work will be done mainly at IC, but decisions as to which route to follow will involve all parties taking into account the timescale and academic needs of the student.

大多数毕赤酵母分泌的重组蛋白的比生产率相对较低。高产重组菌株的鉴定在很大程度上是一种经验方法，对导致培养条件从摇瓶规模扩大到高细胞密度工业发酵时的高生产率的特征了解甚少。当使用巴斯德毕赤酵母表达蛋白质时，目前的实践是使用整合载体产生重组体，导致位置非依赖性表达。最广泛使用的系统是基于醇氧化酶启动子（AOX），其需要去阻遏，然后用甲醇诱导。靶基因的拷贝数是一个重要变量，但不一定与滴度相关。用于规模扩大和生产的“最佳”克隆的选择是复杂的，因为在摇瓶中实现高分泌蛋白滴度的克隆在高细胞密度发酵中并不总是实现高滴度。这也受到宿主甲醇利用表型（mut+、mutS或mut-）影响的显著影响。研究（限于实验室过程）表明，在巴斯德毕赤酵母中的表达差在某些情况下可能与ER中的折叠问题相关，这会诱导未折叠蛋白反应（UPR）。BRIC项目06/18（大卫莱克，帝国理工学院（IC））是（1）开发代谢组学方法，并将其与转录组学一起应用，以了解UPR应激、非应激和非表达培养物的潜在生理学，（2）开发易于监测的UPR报告基因，以允许在小规模上确定理想的表达条件。该项目将使用工业相关蛋白质（用甲醇和非甲醇诱导）和蛋白质模型（低和高分泌滴度）与工业发酵平台，以验证和更好地理解上述参数与UPR诱导之间的相互作用（由BRIC 06/18开发的UPR工具）。还将收集代谢组学信息（基于细胞外（足迹）代谢物）。学生将花费大量的时间在Avanti工作：2-3模型蛋白，在mut宿主范围内的AOX重组体，在摇瓶培养中建立拷贝数和滴度，为进一步工作建立克隆的研究细胞库（mut/拷贝数/滴度矩阵），在15 L补料分批发酵中建立4-6个克隆的发酵性能-包括分析足迹代谢物和/或UPR连接的GFP表达。上述工作预计需要15个月的时间，学生在IC（见下文）和Avalent花费相同的时间。学生最初将在IC工作（约4个月），熟悉正在开发的UPR报告系统，并将构建必要的结构（例如各种mut背景中的GFP），以整合到Avenida的工作中。前半部分（15-18个月）将定义模式/决策矩阵。根据这一结果，我们将：（1）扩大与GFP的工作，这为我们提供了一个现成的系统，用于变体的分选，使其有利于高通量分析，以获得更根本的了解的原因普遍定期审议。这与在IC（BRIC 06/18）进行的转录组学分析相结合，应该指出一些可以进一步开发以改善分泌和蛋白滴度的宿主特征。或者，（2）研究在线控制UPR的可能性，这将需要采用一个/多个表达差的构建体并剖析表达条件以建立比生产率稳定/下降的条件，然后研究我们是否可以开发保持在该水平的反馈控制方案。第二阶段的工作将主要在IC完成，但关于遵循哪条路线的决定将涉及考虑到学生的时间尺度和学术需求的所有各方。