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

该项目涉及的主要研究挑战是提高英国生产重组生物制剂(生物制药)的能力，如抗体片段、生长因子、激素和其他由活细胞生产的基于生物的药物。据估计，重组生物制药的市场年销售额超过1000亿美元。预计到2015年将超过1600亿美元。抗体和抗体碎片的销售在这些销售中占很大比例，这是该领域增长最快的市场。目前获得许可的蛋白质中有三分之一以上是在大肠杆菌中产生的，在这种情况下，从细胞质“出口”到周质是一种受欢迎的策略。这种方法最大限度地降低了下游加工(DSP)的成本，因为(I)可以从相对简单的周质内容物中提纯目标蛋白质，并且(Ii)这避免了碎片和DNA污染，这是严重的DSP问题。使用大肠杆菌是因为它的遗传延展性、安全记录以及快速生长大型和密集培养物的能力。当以大肠杆菌为基础的系统工作良好时，它们可以生产0.5-5g蛋白质/升培养物；然而，目前的大肠杆菌生产平台在过去十年中基本上没有变化，并开始在一些领域达到极限，特别是在生产具有挑战性的折叠或组装路径的生物制药方面。许多产品要么在细胞质中形成不可溶的包涵体(回收率可低至10%)，要么无法出口到周质，因为标准的出口方法只能在未折叠状态下出口蛋白质。在这个项目中，我们的目标是开发改进的大肠杆菌生产系统，它将能够产生前所未有的靶细胞，同时提供非常高的异质性和高折叠完整性的产品。我们将通过专注于上游生产的3个关键领域，并对每个领域的已知问题应用创新解决方案来实现这一纯度的提高。每一个单独部分的成功本身将提高数字信号处理器馈送的质量，而合作伙伴之间的协同作用将导致开发一个整合了所有三项创新的综合平台。转录控制：目前的大肠杆菌生产平台在过去十年中基本上没有变化，依赖于相对较少的启动子。转录控制方面的最新发现将被整合到大肠杆菌中，以大大改善对生物治疗生产的控制，减少导致错误折叠和聚集的生产过剩等问题。这些新的结构，在最先进的组学数据的支持下，也将为生产那些被证明不适合在E.Coli2生产的产品提供新的路线。感知蛋白质折叠：TAT分泌系统输出折叠的蛋白质，从而提供一种将一系列新的产品分泌到周质中的方法。我们将培育出口一系列生物药物的高产优质大肠杆菌菌株。苯乙烯马来酸(SMA)共聚物为周质蛋白提供了更特异和更有效的释放系统，产生了细胞质污染物含量较低的饲料。这种方法提供了一种强大的新方法来释放已出口到外周的生物药物。在整个项目中，我们将与行业合作伙伴合作，确保菌株得到验证并适合用途。总而言之，我们将为行业提供三项关键创新，每一项创新本身都是强大的。同样重要的是，该财团将结合这些创新，创建一条具有独特能力的全新生产线。

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.