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

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 ...