Development of new-generation bacterial secretion process platforms

许多重要的治疗产品是蛋白质--通常被称为生物制药--必须在活的有机体中产生，然后进行纯化。目前获得许可的治疗蛋白中有30%以上是在大肠杆菌中制造的，这种细菌可以快速大量生长。其中一些蛋白质是在细胞内部(细胞质)合成的，但一个受欢迎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。原因有两个。首先，通过选择性地破裂外膜，可以相对容易地提取周质内容物。其次，周质是一个氧化的环境，因此是自然形成二硫键的唯一地方。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‘分泌物’(SEC)途径将蛋白质产物输出到周质中。这个系统以未折叠的状态通过内膜运输蛋白质，之后蛋白质在周质中重新折叠。这通常效果很好，但该系统有严重的局限性：一些蛋白质折叠得太快，SEC系统无法处理，另一些蛋白质可能在周质中无法正确折叠，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。这项应用旨在开发另一种细菌蛋白输出途径，称为TAT途径。这也可以出口外来蛋白质，但主要的区别是它以折叠状态运输蛋白质。重要的是，它似乎只在正确折叠的状态下运输蛋白质，因此它提供了(I)输出SEC途径无法处理的蛋白质，以及(Ii)产生特别高质量的产品，因为它们应该正确折叠和Hance活性。在之前的一个项目中，我们证明了过度表达TAT的大肠杆菌菌株可以非常高的速率输出测试蛋白质--很容易就足以用于工业应用。该项目旨在开发这些菌株的两个重要变种，每个变种都具有独特的特性。该项目将涉及华威和伦敦大学学院之间的合作。这种合作关系很重要：华威团队在TAT研究方面经验丰富，而伦敦大学学院的合作伙伴能够严格测试菌株的质量以及它们是否准备好供行业使用。该项目的第一部分将创造能够输出二硫键结合的预折叠蛋白质的菌株。二硫键通常只在细胞质周围形成，但芬兰的一个小组已经开发出新的大肠杆菌菌株，这种菌株表达一种硫醇氧化酶，使细胞质中有效的二硫键形成。最近的合作研究表明，如果连接一个信号肽，TAT可以有效地输出三个二硫键结合的测试蛋白。这些菌株提供了一种高质量生产二硫键结合蛋白的新方法，有可能产生一种具有非凡折叠保真度的产品。该项目的第二部分旨在利用申请者小组最近的一项令人惊讶的发现。大肠杆菌TAT途径通常将蛋白质输出到周质，外膜在发酵过程中几乎总是保持不变。我们已经用枯草芽孢杆菌的TAT系统(TatAdCd；专利申请)取代了原有的E.ColiTAT系统，并表明该系统还可以高效地将蛋白质输出到周质中。然而，在发酵过程中，外膜变得选择性地渗漏，并将周质蛋白释放到细胞外培养基中(‘肉汤’)。最终结果是，即使在简单的分批发酵中，发酵液中也含有高水平的蛋白质产品，这意味着可以直接从该发酵液中收获产品，而不需要提取周质。这可能是一种非常具有成本效益的生产治疗性蛋白质的新方法。

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.