Exploitation of Tat export machinery for protein production by bacteria

利用 Tat 出口机器通过细菌生产蛋白质

• 批准号: BB/G010501/1
• 负责人: Eli Keshavarz -Moore
• 金额: $ 42.19万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG010501%2F1
• 关键词: Exploitation; Tat; export; machinery; protein

Numerous therapeutic proteins (biopharmaceuticals) are currently produced in bacteria by the biotechnology industry, and this form of production has been in constant use for over 20 years. The Gram-negative bacterium Escherichia coli is the usual host organism for these processes. In some cases the proteins are made in the interior cytoplasm of the bacteria and then extracted, but a favoured approach is to ensure that the proteins of interest are exported out of the cytoplasm into the periplasmic space between the two membranes surrounding the E. coli cell. Once here, the proteins can be purified with relative ease by selectively rupturing the outer membrane. This process has almost always involved exploiting the so-called secretory pathway for protein export, in which the substrate protein is 'threaded' through a pore in the inner membrane in an unfolded state. However, a major problem is that many foreign proteins simply cannot be transported in this manner - they fold too rapidly or tightly before they can be transported. We and others have recently shown that a second protein export system operates in the bacterial inner membrane: the twin-arginine translocation (Tat) system. This system exports proteins that bear transient peptide signals and the Principal Applicant's group has shown that foreign proteins can be efficiently exported by this system. Crucially, the system exports pre-folded proteins. The Tat system thus has huge potential as a platform for the bacterial production of the many recombinant proteins that cannot be exported by traditional means because of folding problems. The main aim of this project is to systematically engineer and analyse E. coli strains that export proteins via the Tat pathway at the high rates demanded by industry. In a concerted effort, the Warwick group will carry out strain improvement and the UCL group will rigorously test the fitness of these strains for industrial use; this is viewed as vital because industrial fermentation systems demand the use of strains that are not prone to lysis or other stress damage. Key elements of the proposal are to (i) identify the most efficient Tat-specific targeting signals, (ii) test the ability of the Tat system to export a range of foreign proteins, (iii) increase Tat-dependent export capacity by over-expressing tat genes and manipulating the levels of key chaperones and proteases, and (iv) develop methods to assess the fitness of engineered strains through ultra-scale-down methods that accurately mimic industrial process conditions. The final section of the E. coli work will be the generation of strains that exhibit an optimal balance of increased export flux and high cell integrity, by identifying the ideal combination of engineered characteristics. These strains will be suitable for use in industrial production systems. The second overall aim of project is to test the feasibility of exploiting Tat-dependent export for recombinant protein production in Gram-positive bacteria. These organisms are not extensively used for production of bipharmaceuticals, but are heavily used for the production of industrial enzymes. Once again, the protein products are ideally exported out of the cell to separate them from the cytoplasmic compartment, but the absence of an outer membrane means that the products are secreted into the growth medium and then purified (there is no periplasmic compartment). The Tat system has real longer-term potential for the production of many recombinant proteins in Gram-positive hosts, and we propose to carry out a feasibility to study to directly assess its potential in the Gram-positive species Bacillus subtilis.

许多治疗性蛋白质（生物药物）目前通过生物技术工业在细菌中生产，并且这种生产形式已经持续使用了20多年。革兰氏阴性细菌大肠杆菌是这些过程的常见宿主生物。在某些情况下，蛋白质是在细菌的内部细胞质中产生的，然后提取出来，但是一种有利的方法是确保感兴趣的蛋白质从细胞质中输出到围绕E的两个膜之间的周质空间。coli细胞。一旦到了这里，蛋白质就可以通过选择性地破坏外膜而相对容易地纯化。这个过程几乎总是涉及利用所谓的分泌途径进行蛋白质输出，其中底物蛋白质以未折叠状态“穿过"内膜中的孔。然而，一个主要的问题是，许多外源蛋白根本不能以这种方式运输-它们在运输之前折叠得太快或太紧。我们和其他人最近发现，第二个蛋白质输出系统在细菌内膜中运作：双精氨酸易位（达特）系统。该系统输出携带瞬时肽信号的蛋白质，并且主要申请人的小组已经表明，外源蛋白质可以通过该系统有效地输出。至关重要的是，该系统输出预折叠的蛋白质。因此，达特系统具有作为细菌生产许多重组蛋白的平台的巨大潜力，这些重组蛋白由于折叠问题而不能通过传统手段输出。本课题的主要目的是对E.通过达特途径以工业所需的高速率输出蛋白质的大肠杆菌菌株。在共同努力下，沃里克小组将进行菌株改良，UCL小组将严格测试这些菌株的工业用途适应性;这被认为是至关重要的，因为工业发酵系统要求使用不易于裂解或其他应激损伤的菌株。该提议的关键要素是（i）鉴定最有效的Tat特异性靶向信号，（ii）测试达特系统输出一系列外源蛋白的能力，（iii）通过过表达tat基因和操纵关键分子伴侣和蛋白酶的水平来增加Tat依赖性输出能力，以及（iv）开发通过精确模拟工业过程条件的超规模缩小方法来评估工程菌株的适应性的方法。E.大肠杆菌的工作将是通过鉴定工程特性的理想组合，产生表现出增加的输出通量和高细胞完整性的最佳平衡的菌株。这些菌株将适用于工业生产系统。项目的第二个总体目标是测试利用Tat依赖性输出在革兰氏阳性菌中生产重组蛋白的可行性。这些生物体不广泛用于生产生物制药，但大量用于生产工业酶。同样，蛋白质产物理想地输出到细胞外以将它们与细胞质区室分离，但是不存在外膜意味着产物分泌到生长培养基中，然后纯化（没有周质区室）。达特系统具有真实的长期潜力，可在革兰氏阳性宿主中生产许多重组蛋白，我们建议进行可行性研究，以直接评估其在革兰氏阳性菌种枯草芽孢杆菌中的潜力。