Investigations of protein ligase for biochemical modification

Ma, Muge 2023. Investigations of protein ligase for biochemical modification. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Asparaginyl endopeptidases (AEPs) are cysteine proteases which are usually found in the vacuolar compartment of plants, they perform transpeptidase function to synthesize cyclotides. AEPs are substrate-specific enzymes requiring a relatively short recognition motif, and they exhibit excellent kinetic properties for peptide and protein ligation. The AEP from plant Oldenlandia affinis (OaAEP1) is the object studied in this thesis, the recombinant OaAEP1 utilized in lab is an engineered mutant, OaAEP1-C247A. However, AEPs have restrictions and challenges in their production and application. This thesis focuses on the development of a superior OaAEP1-C47A variant and the expansion of its application areas in protein modifications. The typical preparation protocol of recombinant OaAEP1-C247A involves complicated procedures which are wasteful in time and workforce. The active AEP generated using the current protocol had the low quality and output that are also tough challenges. To address these problems, a new OaAEP1 construct and its corresponding new preparation protocol with simplified procedures were established and described in Chapter 2. OaAEP1-C247A is a widespread tool of protein backbone modification via site-specific labelling, but limited to the terminus of a substrate. In order to extend the application of AEP, the active AEP produced in Chapter 2 participates in the exploration of protein internal backbone modification by inserting a label into a specific loop region. To achieve the loop insertion into the protein, the corporation of a second transpeptidase is necessary. The protein substrates with exposed internal loops and stable conformations that exhibit high tolerance to the loop opening are ideal model substrate for loop insertion study. The preparation of the other enzyme and model protein substrates is reported in Chapter 3. The loop insertion reaction of protein was completed in sequential model using dual enzymes, and an in-between purification step was involved. To obtain III good yield of final target loop insertion product, reaction conditions of each step were optimized. Additionally, two AEP reversibility inhibition strategies effective in terminal labelling for product yield enhancement were attempted. In Chapter 3, the mechanism and results of loop insertion reaction were described.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Chemistry
Date of First Compliant Deposit:	21 December 2023
Last Modified:	21 Dec 2023 09:05
URI:	https://orca.cardiff.ac.uk/id/eprint/165016

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