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

生物药物是由蛋白质等相对复杂的分子组成的一类药物产品。胰岛素就是生物制药的一个例子。许多生物制药产品，包括胰岛素，都是在大肠杆菌中制造的，因为它有悠久的安全使用历史，具有良好的生理特性，而且相对简单。大肠杆菌可以进行的翻译后修饰的范围是有限的，但蛋白质的二硫键是可能的。为了做到这一点，蛋白质必须被转移到大肠杆菌的周质中，那里是二硫键形成机制(DSB酶)的所在地。移位到周质也可以帮助折叠，保护蛋白质不被蛋白质降解，并简化下游加工。在大肠杆菌中存在三种周质转位途径：TAT、SecB和SRP。TAT途径将折叠的蛋白质从细胞质转移到周质。这条途径最近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条途径SRP和SecB转移到周质，这两条途径都使用SecYEG蛋白复合体将未折叠的重组蛋白运输到周质，在那里折叠。SecB在翻译后转移多肽链，而SRP则通过共翻译转移。通过这三条途径中的任何一条将蛋白质导向周质需要蛋白质的N端有一个信号肽，该信号肽与易位装置结合。在转位过程中，这种信号肽被一种蛋白酶从蛋白质链上切下。因此，信号肽是确定蛋白质到达周质的途径的工具。这项工作的目的是识别和优化能够有效地将选定的重组蛋白运输到周质的信号肽序列。

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.