N-Terminally tagging a substrate for ClpA-mediated unfolding

Bayston, Malachy (2025) N-Terminally tagging a substrate for ClpA-mediated unfolding. Bachelor's Thesis, Biology.

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Abstract

ClpA is a bacterial AAA+ unfoldase that recognises and unfolds substrates tagged with degradation signals. It has been proposed as a candidate for its use the upcoming nanopore-based protein sequencing technology due to potentially having more consistent unfolding steps compared to ClpX. Previous work has highlighted ClpA’s ability to degrade substrates bearing an N-terminal RepA tag. In this project we expressed and purified a RepA-mNG fusion protein and tested its degradation by various ClpA constructs. Our results showed that although full-length RepA�mNG could be purified using a SUMO-tag strategy, degradation remained low in fluorescence assays. While N-terminal RepA tagging shows potential, mNG may be a limiting factor in the assay. Future work should explore GFP as an alternative reporter to improve fluorescence degradation rates and evaluate degradation under high-salt concentrations to better mimic the conditions required in electrophysiology experiments. These experiments could help determine whether ClpA is a suitable unfoldase for nanopore protein sequencing applications.

Item Type:	Thesis (Bachelor's Thesis)
Supervisor name:	Sultanji, S. and Maglia, G.
Degree programme:	Biology
Thesis type:	Bachelor's Thesis
Language:	English
Date Deposited:	02 Jul 2025 10:23
Last Modified:	09 Jul 2025 11:06
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/35523

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