A new generation of E. coli expression hosts and tools for recombinant protein production

该项目所面临的主要研究挑战是提高联合王国生产重组生物制剂（生物药品）的能力，如抗体片段、生长因子、激素和其他用活细胞生产的生物药品。重组生物制药的市场估计每年超过1000亿美元。预计到2015年将超过1600亿美元。抗体和抗体片段的销售占这些销售的很大比例，这是该领域增长最快的市场。目前，超过三分之一的蛋白质是在E。在大肠杆菌中，从细胞质“输出”到周质是一种受欢迎的策略。这种方法使下游处理（DSP）成本最小化，因为（i）可以从相对简单的周质内容物纯化靶蛋白，并且（ii）这避免了作为严重DSP问题的碎片和DNA污染。E.使用大肠杆菌是因为它的遗传可塑性、安全记录和快速生长大而密集的培养物的能力。当E.基于大肠杆菌的系统工作良好，它们可以产生0.5 - 5g蛋白质/升培养物;大肠杆菌生产平台在过去十年中基本上没有变化，并且在许多领域开始达到其极限，特别是在具有挑战性折叠或组装途径的生物药物的生产中。许多产物要么在细胞质中形成不溶性包涵体（回收率可低至10%），要么不能输出到周质，因为标准输出方法只能输出未折叠状态的蛋白质。大肠杆菌生产系统，将能够产生前所未有的范围的目标分子，同时提供非常高的“质量”的产品，在最小的异质性和高折叠完整性。我们将通过专注于上游生产的3个关键领域并针对每个领域的已知问题应用创新解决方案来实现纯度的提高。每个单独部分的成功将提高DSP饲料的质量，而合作伙伴之间的协同作用将导致开发一个集成了所有3项创新的集成平台。转录调控：现行E.大肠杆菌生产平台在过去十年中基本上没有变化，依赖于相对少量的启动子。转录调控的最新发现将被纳入E.大肠杆菌中，以允许大大改善的双链体生产的控制，减少问题，如过度生产，导致错误折叠和聚集。这些新的构建体，在最新的组学数据的支持下，也将为生产那些已经被证明在E. coli.2.传感蛋白折叠：达特分泌系统输出折叠蛋白，从而提供了一种将新范围的产物分泌到周质中的方法。我们将开发E。大肠杆菌菌株，输出一系列生物制药高产和产品质量.苯乙烯马来酸（SMA）共聚物为周质蛋白提供更特异和有效的释放系统，产生细胞质污染物低的饲料。这种方法提供了一种强有力的新手段，释放已输出到周质的生物药物。在整个项目过程中，我们将与工业合作伙伴合作，以确保菌株得到验证并符合目的。总之，我们将为工业提供三项关键创新，每一项都是强大的。同样重要的是，该财团将联合收割机结合这些创新，创造一个全新的生产管道，具有独特的能力。

The main research challenge addressed in this project is to enhance the UK's capacity for the production of recombinant biologics (biopharmaceuticals) such as antibody fragments, growth factors, hormones and other biologically-based medicines produced from live cells. The market for recombinant biopharmaceuticals is estimated to be over $100 billion p.a. and is predicted to exceed $160 billion by 2015. Sales of antibodies and antibody fragments account for a large proportion of these sales and this is the fastest-growing market in this sector. Over a third of currently-licensed proteins are produced in E. coli, where 'export' out of the cytoplasm to the periplasm is a favoured strategy. This approach minimises downstream processing (DSP) costs because (i) the target protein can be purified from the relatively simple periplasmic contents, and (ii) this avoids debris and DNA contamination which are serious DSP problems. E. coli is used because of its genetic malleability, safety record and the ability to rapidly grow large and dense cultures. When E. coli-based systems work well, they can produce 0.5 - 5g protein/litre culture; however, current E. coli production platforms have been largely unchanged for the last decade and are beginning to reach their limits in a number of areas, especially in the production of of biopharmaceuticals that have challenging folding or assembly pathways. Many products either form insoluble inclusion bodies in the cytoplasm (where recoveries can be as low as 10%) or fail to be exported to the periplasm because the standard export method is only capable of exporting proteins in an unfolded state.In this project we aim to develop improved E. coli production systems that will be capable of producing an unprecedented range of target mocules, while delivering products of very high 'quality' in terms of minimal heterogeneity and high folding integrity. We will achieve this increase in purity by focusing on 3 key areas of upstream production and applying innovative solutions to known problems in each area. Success in each individual section on its own will enhance the quality of DSP feed, while synergies between partners will lead to the development of an integrated platform that incorporates all 3 innovations.1. Transcriptional control: current E. coli production platforms have been largely unchanged for the last decade relying on a relatively small number of promoters. The latest discoveries in transcriptional control will be incorporated into E. coli to allow much-improved control of biotherapeutic production, reducing problems such as overproduction which leads to mis-folding and aggregation. These new constructs, backed up by state of the art 'omics data, will also provide new routes for producing those products that have proved to be recalcitrant to production in E. coli.2. Sensing protein folding: the Tat secretion system exports folded proteins and thereby provides a method for secreting a new range of products into the periplasm. We will develop E. coli strains that export a range of biopharmaceuticals with high yield and product quality.3. Styrene Maleic Acid (SMA) co-polymer provides a more specific and efficient release system for periplasmic proteins, yielding a feed that is low in cytoplasmic contaminants. This method provides a powerful new means of releasing biopharmaceuticals that have been exported to the periplasm. Throughout the project we will work with industrial collaborators to ensure that the strains are validated and fit for purpose.IN SUMMARY, we will provide industry with three key innovations, each of which is powerful in its own right. Equally importantly, the consortium will combine these innovations to create a wholly-novel production pipeline with unique capabilities.