Development of new-generation bacterial secretion process platforms

新一代细菌分泌过程平台开发

• 批准号: BB/K011219/1
• 负责人: Colin Robinson
• 金额: $ 45.4万
• 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: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK011219%2F1
• 关键词: Development; new; generation; bacterial; secretion

Many important therapeutic products are proteins - often termed biopharmaceuticals - that have to be produced in a living organism and then purified. Over 30% of the currently licensed therapeutic proteins are made in the bacterium Escherichia coli, which can be quickly grown in large amounts. Some of these proteins are synthesised in the cell interior (cytoplasm) but a favoured strategy is to 'export' the protein product to the periplasmic space between the two cell membranes. The reasons are two-fold. First, the contents of the periplasm can be extracted relatively easily, by selectively rupturing the outer membrane. Secondly, the periplasm is an oxidising environment, and is thus the only place where disulphide bonds form naturally. These bonds are essential structural features of some proteins. Industrial applications almost always use the bacterial 'secretory' (Sec) pathway to export the protein product to the periplasm. This system transports the protein through the inner membrane in an unfolded state, after which the protein refolds in the periplasm. This often works very well but the system has serious limitations: some proteins fold too quickly for the Sec system to handle, and others may not fold correctly in the periplasm, which lacks the natural 'chaperone' molecules that normally help most proteins to fold in the cytoplasm. This application aims to exploit a second bacterial protein export pathway, known as the Tat pathway. This can also export foreign proteins, but the major difference is that it transports proteins in a folded state. Importantly, it appears only to transport proteins in a correctly-folded state, and it therefore offers potential for (i) exporting proteins that the Sec pathway cannot handle, and (ii) producing products of particularly high quality, since they should be correctly folded and hance active. In a previous project, we showed that E. coli strains over-expressing Tat could export a test protein at very high rates - easily sufficient for industrial applications. This project aims to develop two important variants of these strains, each with unique properties. The project will involve collaboration between Warwick and UCL. The partnership is important: the Warwick group are experienced in Tat studies while the UCL partner is able to rigorously test the quality of strains and their readiness for use by industry. The first part of the project will create strains that can export prefolded proteins that are disulphide-bonded. Disulphide bonds normally only form in the periplasm, but a Finnish group has developed new E. coli strains which express a thiol oxidase that enables efficient disulphide bond formation in the cytoplasm. Recent collaborative studies have shown that three disulphide-bonded test proteins are efficiently exported by Tat if a signal peptide is attached. These strains offer a new means of producing disulphide-bonded proteins in high quantities, with the potential of generating a product of exceptional folding fidelity.The second part of the project aims to exploit a surprising recent finding by the applicants' groups. The E. coli Tat pathway normally exports proteins to the periplasm, and the outer membrane almost invariably remains intact during fermentation processes. We have replaced the native E. coli Tat system with a Tat system from Bacillus subtilis (TatAdCd; patent application filed) and have shown that the system also exports proteins to the periplasm with high efficiency. However, during fermentation the outer membrane becomes selectively leaky, and releases periplasmic proteins into the extracellular medium ('broth'). The net result is that even in simple batch fermentations, the broth contains high levels of the protein product and this means that the product can be harvested directly from this broth without the need for extraction of the periplasm. This may be a very cost-effective new means of producing therapeutic proteins.

许多重要的治疗产品是蛋白质-通常称为生物药物-必须在活的有机体中产生，然后纯化。目前超过30%的治疗性蛋白质是在大肠杆菌中生产的，这种细菌可以大量快速生长。这些蛋白质中的一些在细胞内部（细胞质）合成，但优选的策略是将蛋白质产物“输出”到两个细胞膜之间的周质空间。原因有两方面。首先，通过选择性地破坏外膜，可以相对容易地提取周质的内容物。其次，周质是一个氧化环境，因此是唯一的地方，二硫键自然形成。这些键是某些蛋白质的基本结构特征。工业应用几乎总是使用细菌“分泌”（Sec）途径将蛋白质产物输出到周质。该系统将蛋白质以未折叠状态运输通过内膜，之后蛋白质在周质中重新折叠。这通常工作得很好，但该系统有严重的局限性：一些蛋白质折叠太快，Sec系统无法处理，而其他蛋白质可能无法在周质中正确折叠，周质缺乏通常帮助大多数蛋白质在细胞质中折叠的天然“伴侣”分子。本申请旨在开发第二种细菌蛋白质输出途径，称为达特途径。这也可以输出外源蛋白质，但主要区别在于它以折叠状态运输蛋白质。重要的是，它似乎只运输正确折叠状态的蛋白质，因此它提供了（i）输出Sec途径无法处理的蛋白质，以及（ii）生产特别高质量的产品的潜力，因为它们应该正确折叠并具有高活性。在以前的一个项目中，我们表明，E。过表达达特的大肠杆菌菌株可以以非常高的速率输出测试蛋白-容易地足以用于工业应用。该项目旨在开发这些菌株的两种重要变体，每种变体都具有独特的特性。该项目将涉及沃里克和伦敦大学学院之间的合作。伙伴关系很重要：沃里克集团在达特研究方面经验丰富，而UCL合作伙伴能够严格测试菌株的质量及其在工业上的使用准备。该项目的第一部分将创建可以输出二硫键结合的预折叠蛋白质的菌株。二硫键通常只在周质中形成，但芬兰的一个研究小组已经开发出新的E。大肠杆菌菌株，其表达能够在细胞质中有效形成二硫键的硫醇氧化酶。最近的合作研究表明，三个二硫键键合的测试蛋白质被有效地输出的达特，如果一个信号肽连接。这些菌株提供了大量生产二硫键结合蛋白质的新方法，具有产生具有特殊折叠保真度的产物的潜力。该项目的第二部分旨在利用申请人小组最近的一项令人惊讶的发现。急诊大肠杆菌达特途径通常将蛋白质输出到周质，并且外膜在发酵过程中几乎总是保持完整。我们已经取代了本土的E。coli达特系统与来自枯草芽孢杆菌的达特系统（TatAdCd;已提交专利申请）的比较，并且已经显示该系统也以高效率将蛋白质输出到周质。然而，在发酵过程中，外膜变得选择性渗漏，并将周质蛋白释放到细胞外培养基（“肉汤”）中。最终结果是，即使在简单的分批发酵中，发酵液也含有高水平的蛋白质产物，这意味着可以直接从该发酵液中收获产物，而不需要提取周质。这可能是生产治疗性蛋白质的一种非常具有成本效益的新方法。

项目成果

The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity

枯草芽孢杆菌 TatAdCd 系统表现出极高水平的底物选择性

• DOI: 10.1016/j.bbamcr.2016.10.018
• 发表时间: 2017
• 期刊: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
• 作者: Frain K

Exclusively membrane-inserted state of an uncleavable Tat precursor protein suggests lateral transfer into the bilayer from the translocon.

不可切割的 Tat 前体蛋白的完全膜插入状态表明从易位子横向转移到双层中。

• DOI: 10.1111/febs.12327
• 发表时间: 2013
• 期刊: The FEBS journal
• 作者: Ren C

High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization.

• DOI: 10.1002/btpr.2534
• 发表时间: 2018-01
• 期刊: Biotechnology progress
• 影响因子: 2.9
• 作者: Velez-Suberbie ML;Betts JPJ;Walker KL;Robinson C;Zoro B;Keshavarz-Moore E
• 通讯作者: Keshavarz-Moore E

Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli.

在大肠杆菌中生产单跨膜蛋白的新方法的表征。

• DOI: 10.1002/bit.26895
• 发表时间: 2019
• 期刊: Biotechnology and bioengineering
• 影响因子: 3.8
• 作者: Smith SM