The effect of signal peptide sequence on transcription, translation and translocation of recombinant proteins to the periplasm of E. coli.

信号肽序列对重组蛋白转录、翻译和易位至大肠杆菌周质的影响。

• 批准号: 2098552
• 金额: --
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2098552
• 关键词: effect; signal; peptide; sequence; transcription

Biopharmaceuticals are a class of drug products comprising relatively complex molecules such as proteins. An example biopharmaceutical is insulin. Many biopharmaceutical products, including insulin, are made in E. coli because of its long history of safe use, well-characterised physiology and relative simplicity. The range of post-translational modifications that E. coli can perform is limited, but disulphide bonding of proteins is possible. In order to do this, proteins must be translocated to the periplasm of E. coli where the disulphide bond formation machinery (the Dsb enzymes) reside. Translocation to the periplasm can also aid folding, protect proteins from proteolysis, and simplifies downstream processing. There are three periplasmic translocation pathways in E. coli: Tat; SecB; and SRP. The Tat pathway translocates folded proteins from the cytoplasm to the periplasm. This pathway has recently been developed for generating recombinant protein. However, the majority of recombinant proteins are translocated to the periplasm by the two other pathways, SRP and SecB, both of which use the SecYEG protein complex to transport the unfolded recombinant protein to the periplasm, where it folds. SecB translocates polypeptides chains following translation, whereas SRP translocates co-translationally.Directing the protein to the periplasm through any of these three pathways requires a signal peptide on the N terminus of the protein, which binds to the translocation apparatus. This signal peptide is cut from the protein chain by a protease during translocation. The signal peptide is therefore the tool that identifies the pathway the protein utilises to reach the periplasm.This work aims to identify and optimise the signal peptide sequence that can effectively transport selected recombinant proteins to the periplasm.

生物药物是一类包含相对复杂的分子如蛋白质的药物产品。生物制药的一个例子是胰岛素。许多生物制药产品，包括胰岛素，都是在大肠杆菌中生产的。大肠杆菌，因为它的安全使用，良好的生理特征和相对简单的历史。E.大肠杆菌可以执行的是有限的，但蛋白质的二硫键是可能的。为了做到这一点，蛋白质必须转移到E。二硫键形成机器（Dsb酶）所在的大肠杆菌。易位到周质也可以帮助折叠，保护蛋白质免受蛋白水解，并简化下游加工。E. coli：达特、SecB和SRP。达特途径将折叠的蛋白质从细胞质易位到周质。该途径最近已被开发用于产生重组蛋白。然而，大多数重组蛋白通过另外两种途径（SRP和SecB）转运到周质，这两种途径都使用SecYEG蛋白复合物将未折叠的重组蛋白转运到周质，并在周质中折叠。SecB在翻译后转位多肽链，而SRP在翻译后转位多肽链，通过这三条途径中的任何一条将蛋白质引导至周质需要蛋白质N末端的信号肽，该信号肽结合到转位装置。该信号肽在移位过程中被蛋白酶从蛋白链上切割下来。因此，信号肽是识别蛋白质利用到达周质的途径的工具。这项工作的目的是识别和优化信号肽序列，可以有效地将选定的重组蛋白质运输到周质。