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

Lead Research Organisation: University of Birmingham
Department Name: Chemical Engineering

Abstract

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.

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
2098552 Studentship BB/M01116X/1 01/10/2018 30/09/2022 Parisa Pordelkhaki