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

生物制药是一类药物产品，包括相对复杂的分子，如蛋白质。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，都是在大肠杆菌中制造的，因为它的安全使用历史悠久，生理特征明确，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外质中，那里有二硫键形成机制（Dsb酶）。转运到外周质也可以帮助折叠，保护蛋白质免于蛋白质水解，并简化下游加工。大肠杆菌有三种质周易位途径：Tat；SecB;和SRP。Tat途径将折叠蛋白从细胞质转运到外周质。该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种途径转运到外周质，即SRP和SecB，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外周质，并在那里折叠。SecB在翻译后易位多肽链，而SRP在翻译后易位。通过这三种途径中的任何一种将蛋白质引导到外周质都需要蛋白质N端上的信号肽，该信号肽与易位装置结合。这种信号肽在转运过程中被蛋白酶从蛋白质链上切下。因此，信号肽是识别蛋白质到达外周质途径的工具。本工作旨在鉴定和优化信号肽序列，以有效地将选择的重组蛋白转运到外质。

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.