UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Biochemical and biophysical characterisation of Thermotoga maritima and Myxococcus xanthus encapsulins towards biotechnology applications

Van De Steen, Alexander; (2023) Biochemical and biophysical characterisation of Thermotoga maritima and Myxococcus xanthus encapsulins towards biotechnology applications. Doctoral thesis (Ph.D), UCL (University College London).

[thumbnail of Correction submitted-copyright.pdf] Text
Correction submitted-copyright.pdf - Submitted Version
Access restricted to UCL open access staff until 19 June 2024.

Download (7MB)

Abstract

As synthetic systems become multi-levelled, spatial control of biomolecules is increasingly important for sub-cellular and in vitro applications. Protein compartments such as encapsulin compartments offer definitive structures with potential for surface scaffolding functions and applications in biocatalysis. However, the use of encapsulin compartments requires detailed knowledge of their biochemical and physical properties and limitations of both the compartment and the encapsulated or displayed cargo. This is the overarching topic of this thesis. Firstly, we explored the compartment stability landscape of two of the most studied encapsulin classes from Thermotoga maritima (T1) and Myxococcus xanthus (T3). We analysed both compartments over a wide pH and temperature range and combined results from intrinsic and extrinsic fluorescent measurements, static and dynamic light scatter, along with Transmission Electron Microscopy, native PolyAcrimade Gel Electrophoresis (PAGE) and Size exclusion chromatography (SEC) to measure compartment integrity over acidic pH and temperature ranges. Both encapsulins show high thermal stability from pH 4.0 to pH 8.0 with aggregation occurring before the loss of quaternary structure. Secondly, the cargo stability was investigated, by loading encapsulins with a GFP variant and an enzyme in vitro, and comparing the stability gain of loaded cargo compared to free. Reassembly was dependent on type of denaturant used, and efficiency of reassembly for M. xanthus compartments was found to be generally poor. Proportional loading with increasing GFP concentrations was observed for T. maritima encapsulins, whereas in M. xanthus GFP cargo displayed a scaffolding function at a low molar ratio of GFP to shell monomer, after which reassembly was reduced. Moving from GFP to application focus, an industrially relevant enzymatic cargo, transketolase homodimer, was encapsulated and retained its active conformation. Stability analysis utilising a FRET system showed a 15 °C increase in thermal melting transition of encapsulated enzyme versus free enzyme, showing a protective effect of encapsulation. Finally, a biomedical proof-of-concept study employed the T. maritima encapsulin as a modular drug delivery system (DDS). Outer surface display of a nanobody mimic Designed Ankyrin Repeat (DARPin 929) specific to the human epidermal growth factor 2 (HER2) receptor showed localisation of the DDS to HER2+ breast cancer cells in cell culture. Combined with E. coli in vivo loading of an engineered flavin binding protein, it was shown that the DDS generated reactive oxygen species in response to blue light, triggering apoptosis in targeted HER2+ cells. In summary, in the project a range of biochemical and biophysical tools were applied to further the understanding of critical stability and integrity parameters of encapsulin compartments and cargo proteins. This understanding underpins the generation of an encapsulin-based enzyme stabilisation particle and a drug delivery system. This thesis will support the further development of encapsulin applications but also wider protein compartment considerations.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Biochemical and biophysical characterisation of Thermotoga maritima and Myxococcus xanthus encapsulins towards biotechnology applications
Language: English
Additional information: Copyright © The Author 2023. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
Keywords: Protein compartments, encapsulin, biotechnology
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10171084
Downloads since deposit
6Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item