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

新一代大肠杆菌表达宿主和重组蛋白生产工具

• 批准号: BB/M018288/1
• 负责人: Colin Robinson
• 金额: $ 99.09万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM018288%2F1
• 关键词: new; generation; E.; coli; expression

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.

该项目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。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。目前获得许可的蛋白质中有超过三分之一是在大肠杆菌中生产的，在大肠杆菌中，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处理（DSP）的成本，因为(i)目标蛋白可以从相对简单的质周内容物中纯化，（ii）这避免了碎片和DNA污染，这是严重的DSP问题。大肠杆菌之所以被使用，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。当大肠杆菌为基础的系统工作良好时，它们可以产生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没有变化，并且在许多领域开始达到极限，特别是在具有挑战性的折叠或组装途径的生物制药生产中。许多产物要么在细胞质中形成不溶性包涵体（回收率低至10%），要么无法输出到外周质，因为标准的输出方法只能输出未折叠状态的蛋白质。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，该系统将能够生产前所未有的目标模块范围，同时在最小的异质性和高折叠完整性方面提供非常高的“质量”产品。我们将专注于上游生产的3个关键领域，并对每个领域的已知问题采用创新的解决方案，以实现纯度的提高。每个单独部分的成功将提高DSP馈送的质量，而合作伙伴之间的协同作用将导致集成平台的发展，包括所有3个创新。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纳入大肠杆菌，从而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，由最先进的组学数据支持，也将为生产那些被证明在大肠杆菌中难以生产的产品提供新的途径。感知蛋白质折叠：Tat分泌系统输出折叠的蛋白质，从而提供了一种向外周质分泌一系列新产品的方法。我们将开发大肠杆菌菌株，以出口一系列高产、高质量的生物制药产品。苯乙烯马来酸（SMA）共聚物为质周蛋白提供了一种更特异、更有效的释放系统，从而产生了一种低细胞质污染物的饲料。该方法为已出口到外周的生物药物的释放提供了一种强有力的新手段。在整个项目中，我们将与工业合作者合作，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个关键的创新，每一个都有自己的强大之处。同样重要的是，该联盟将把这些创新结合起来，创造一个具有独特能力的全新生产管道。

项目成果

Exposure to 1-Butanol Exemplifies the Response of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius to Solvent Stress.

• DOI: 10.1128/aem.02988-20
• 发表时间: 2021-05-11
• 期刊: Applied and environmental microbiology
• 影响因子: 4.4
• 作者: Benninghoff JC;Kuschmierz L;Zhou X;Albersmeier A;Pham TK;Busche T;Wright PC;Kalinowski J;Makarova KS;Bräsen C;Flemming HC;Wingender J;Siebers B
• 通讯作者: Siebers B

Novel constructs and 1-step chromatography protocols for the production of Porcine Circovirus 2d (PCV2d) and Circovirus 3 (PCV3) subunit vaccine candidates

• DOI: 10.1016/j.fbp.2021.10.001
• 发表时间: 2021-11-25
• 期刊: FOOD AND BIOPRODUCTS PROCESSING
• 影响因子: 4.6
• 作者: Peswani, Amber R.;Narkpuk, Jaraspim;Robinson, Colin
• 通讯作者: Robinson, Colin

Escherichia coli "TatExpress" strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway.

• DOI: 10.1002/bit.26434
• 发表时间: 2017-12
• 期刊: Biotechnology and bioengineering
• 影响因子: 3.8
• 作者: Browning DF;Richards KL;Peswani AR;Roobol J;Busby SJW;Robinson C
• 通讯作者: Robinson C