Using GFP to investigate protein localisation, function and global cellular response

Abstract

Integral membrane proteins (IMPs) make up 20-30 % of genes in all walks of life. They are major determinants of disease pathology, making them prime therapeutic targets for cancer, bacterial infection and genetic disorders. Despite this, they are underrepresented in the literature; this is often attributed to complications with IMP expression, purification and characterisation. This thesis aimed to tackle the difficulties with IMP characterisation. Exploitation of tagged versions of the IMPs has opened up new research fields, initially based on simple observations. The tag used is the well characterised GFP which has allowed for the simple optimisation of conditions for protein over-expression, determination of intracellular localisation of H. sapiens SWEET sugar transporter, development of a ligand binding method that does not rely on properties of the ligand and demonstrated that protein over-expression using the Escherichia coli pET system only occurs in mutant forms of this organism. The implication being that protein over-expression only occurs via genetically modified organisms.

Ubiquitously expressed, the MFS is one of the largest protein superfamilies, with roles including metabolite and xenobiotic transport. They have been implicated in the development of antimicrobial resistance in E. coli, making them a clinically relevant target. An expression library of 63 GFP-tagged proteins was produced, before screening for optimal expression conditions. The larger amounts of transporter obtained via this approach enabled the implementation of a ligand-binding assay using the technique of thermophoresis. This approach produced novel binding substrates as well as identifying a new binding event for the well characterised drug efflux transporter mdfA. Significantly, the approach has shown that cyclic AMP also binds to mdfA and another MFS transporter kgtP, potentially identifying a novel role for these transporters in the catabolite repression process.

The work presented in this thesis has shown the versatility of a reporter system like GFP to uncover fundamental properties at the cellular level (protein localisation experiments), the biochemical level (optimisation of protein over-expression and ligand binding studies) but also at the cellular level (E. coli’s use of mutants during protein over-expression). This research is a building block to identifying new drug targets to tackle the global problem of antimicrobial resistance.

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