Novel Tat-based systems for recombinant protein production and surface display in bacteria

大肠杆菌广泛用于生产治疗用蛋白质。示例产品包括治疗性抗体，胰岛素和许多其他产品，每年的总市场约为1000亿美元。一种优选的方法是将目的蛋白质“输出”到两个细胞膜之间的周质空间，因为蛋白质的纯化通常更容易且更具成本效益。传统上，蛋白质输出是通过“Sec”途径实现的，该途径将重组蛋白以未折叠的形式运输穿过内膜。然而，一些蛋白质产品在运输后不能再折叠，或者在运输前折叠过快，因此不能生产。达特蛋白输出途径提供了Sec途径的替代方法，因为它以预折叠形式运输蛋白底物，这绕过了在运输前解折叠蛋白质和之后重折叠蛋白质所涉及的困难步骤。我们最近首次证明了达特可以输出非常大量的蛋白质。大肠杆菌达特系统输出蛋白质到周质，就像Sec系统一样，但是我们最近创建了一个大肠杆菌Tat系统。大肠杆菌菌株，其表达来自另一种细菌枯草芽孢杆菌的达特系统（称为TatAdCd）来代替天然大肠杆菌。coli达特系统。这种菌株有独特的能力。它通过达特途径有效地将蛋白质产物转运到周质，但随后将产物释放到培养基中，因为它的外膜上有大的孔。细胞在其他方面是健壮的，可以毫无问题地培养。该项目将利用该菌株用于两个目的。首先，我们将开发用于小规模和大规模生产治疗性蛋白质的系统。该菌株具有用于此目的的巨大潜力，因为蛋白质产物可以直接从培养基中纯化，绕过了从周质空间提取的有问题的步骤，并大大简化了下游加工。该项目的这一部分还将涉及使用另一种新技术，涉及硫醇氧化酶的表达，以输出正确折叠的二硫键结合蛋白质。许多蛋白质含有二硫键，巯基氧化酶帮助底物蛋白质在输出前形成二硫键。这将有助于达特系统输出正确折叠的“高质量”蛋白质。其次，我们将开发一种新的系统，用于蛋白质的“表面展示”和相互作用的伴侣蛋白质的分离。许多治疗性蛋白质针对特定的蛋白质靶标，例如癌细胞上的那些，并且分离新的治疗性蛋白质的关键方法是在细菌细胞表面上表达大量不同蛋白质的文库，然后添加标记的靶蛋白。分离表达与靶标结合的蛋白质的细胞，并克隆感兴趣的蛋白质以进一步测试作为治疗性蛋白质。目前的表面展示方法具有真实的局限性，包括要求蛋白质文库首先通过Sec途径转运穿过细胞膜。我们建议开发一个新的系统，使用TatAdCd表达细胞。在该方法中，蛋白质文库将通过达特途径输出并拴系到内膜的外部。由于这些细胞的外膜含有大的孔，添加的标记的靶蛋白将能够结合到暴露的蛋白质和相互作用的伙伴将很容易识别。这项技术应该导致识别重要的新的治疗蛋白质，已经错过了使用目前的技术。

The bacterium Escherichia coli is extensively used to produce proteins for therapeutic use. Example products include therapeutic antibodies, insulin and many others, with a total market of around $100 billion per year. A preferred approach is to 'export' the protein of interest to the periplasmic space between the 2 cell membranes, because purification of the protein is often easier and more cost-effective. Traditionally, protein export is achieved by the 'Sec' pathway, which transports the recombinant protein across the inner membrane in an unfolded form. However, some protein products either cannot refold after transport, or fold too quickly before they are transported, and thus cannot be produced. The Tat protein export pathway offers an alternative approach to the Sec pathway because it transports the protein substrate in a pre-folded form, which bypasses difficult steps involved in unfolding the protein before transport, and refolding it afterwards. We have recently shown for the first time that Tat can export very large quantities of protein.The natural E. coli Tat system exports proteins to the periplasm, just like the Sec system, but we have recently created an E. coli strain that expresses a Tat system (termed TatAdCd) from another bacterium, Bacillus subtilis, in place of the native E. coli Tat system. This strain has unique abilities. It efficiently transports the protein product to the periplasm by the Tat pathway, but then releases the product into the culture medium because it has large holes in its outer membrane. The cells are otherwise robust and can be cultured without problems. This project will use this strain for two purposes.First, we will develop the system for the small- and large-scale production of therapeutic proteins. The strain has enormous potential for this purpose because protein products can be purified directly from the culture medium, bypassing the problematic steps of extraction from the periplasmic space and greatly simplifying downstream processing. This part of the project will also involve use of another new technology, involving expression of a thiol oxidase, in order to export correctly folded disulphide-bonded proteins. Many proteins contain disulphide bonds, and the the thiol oxidase enzyme helps the substrate protein to form disulphide bonds before export. This will help the Tat system to export correctly folded 'high-quality' proteins.Secondly, we will develop a novel system for 'surface display' of proteins and isolation of interacting partner proteins. Many therapeutic proteins are directed against specific protein targets, for example those on cancer cells, and a key method for isolating new therapeutic proteins is to express a massive library of different proteins on the bacterial cell surface and then add labeled target protein. Cells expressing a protein that binds to the target are isolated and the protein of interest is cloned for further testing as a therapeutic protein. Current surface display methods have real limitations, including a requirement that the library of proteins is first transported across the cell membrane by the Sec pathway. We propose to develop a new system using the TatAdCd-expressing cells. In this method, the library of proteins will be exported by the Tat pathway and tethered to the outside of the inner membrane. Because the outer membranes of these cells contain large holes, added labeled target protein will be able to bind to the exposed proteins and interacting partners will be readily identified. This technique should result in the identification of important new therapeutic proteins that have been missed using current techniques.