Solubilisation and stabilisation of MRP4 using novel detergents for function and structural studies

Hardy, David (2019). Solubilisation and stabilisation of MRP4 using novel detergents for function and structural studies. PHD thesis, Aston University.

Abstract

Knowledge of membrane protein function and structure is limited due to the complications in the production of stable, functional membrane proteins that can be used in functional and structural studies. These limitations are due to their location within a lipid bilayer and this creates challenges at almost every stage including expression, solubilisation, stabilisation and purification of membrane proteins. This study aimed to combat some of these limitations for the human membrane protein ABCC4/MRP4 (multidrug resistance protein 4) from the ABC superfamily, in particular focusing on the solubilisation and stabilisation aspects. MRP4 was expressed in Spodoptera frugiperda (Sf 9) insect cells, Pichia pastoris yeast cells and Human Embryonic Kidney (HEK) cells. It was found that the highest level of expression was achieved using Sf 9 insect cells. MRP4 was then tested for functionality using a fluorescent vesicular transport assay that was developed within this study and shown to be functional. It was found that Calixarene C4C7 and the polymer SMA 2000 were both more efficient at solubilising and stabilising MRP4 than conventional detergents. Conditions for purification with both C4C7 and SMA 2000 were optimised, and were able to produce protein at ~40% and ~70% purity and concentrations of ~400 μg/mL and ~25 μg/mL respectively. Following purification it was determined, using tryptophan fluorescence quenching binding assays, that the SMA purified MRP4 bound ligands and retained increased stability compared to conventional detergents. Preliminary studies were performed using electron microscopy, which showed potential for this approach in the future. The techniques and methods developed in this study can be used by others in the future to gain better structural and functional knowledge of MRP4 and can also be applied to the study of other membrane proteins.

Divisions: Life & Health Sciences > Biosciences
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Institution: Aston University
Uncontrolled Keywords: Membrane proteins,expression,purification,Calixar,SMALP
Completed Date: 2019-03-13
Authors: Hardy, David

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