Chemoenzymatic backbone labelling of proteins by ‘loop insertion’

Dickerson, Matthew 2025. Chemoenzymatic backbone labelling of proteins by ‘loop insertion’. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Bioconjugation of proteins is a crucial area in modern biochemistry, enabling the development of novel protein-based tools for a wide range of applications. Current bioconjugation strategies primarily target protein termini or specific side chain functional groups of amino acids. While advances have led to more selective residue-specific labelling, efficient and minimally disruptive labelling of the protein backbone remains a significant challenge. In response to this need, a novel protein backbone labelling strategy, termed ‘loop insertion’, has been developed. This approach combines recent advancements in enzyme-mediated bioconjugation using the asparaginyl ligase OaAEP1 with established chemical methods such as native chemical ligation. To explore the feasibility of loop insertion, the reaction was first evaluated using a simplified peptide model. This model highlighted the importance of ligation directionality and demonstrated that the combination of OaAEP1 and native chemical ligation yielded the most effective results. In order to optimise the yield of loop inserted product. A fully enzymatic method was also trialled, but due to poor pH compatibility between the peptide ligase omniligase and OaAEP1 the chemoenzymatic approach was preferred. Building on this, the loop insertion method was extended to structurally complex protein models, including green fluorescent protein (GFP) and maltose binding protein (MBP), both of which were successfully labelled using the loop insertion strategy. Furthermore, the approach was compatible with the incorporation of both functional and non-canonical peptides into the protein backbone. This novel methodology offers a powerful new tool for site-specific protein backbone modification, expanding the capabilities of protein engineering beyond the limitations of current semi-synthetic or side chain labelling techniques.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Chemistry
Date of First Compliant Deposit:	26 February 2026
Last Modified:	26 Feb 2026 16:31
URI:	https://orca.cardiff.ac.uk/id/eprint/185335

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