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Title: Expression, purification and functional characterisation of the adenosine A2a receptor : producing a protein optimum for structural studies
Author: Singh, Shweta
ISNI:       0000 0004 2684 0710
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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G-protein coupled receptors (GPCRs) are cell surface receptors which interact with a diverse range of external stimuli including hormones, neurotransmitters and many drugs and thereby mediate a wide range of intracellular responses. Their importance in health and disease has made them the subject of intense study, particularly in terms of understanding their structure-function relationships. Adenosine A2aR is a GPCR activated by the nucleoside, adenosine. It is present ubiquitously within the human body and has roles in immune regulation, sleep induction and neurological disorders making it an important target for structural work. The aim of the research outlined in this thesis was to develop reliable, reproducible protocols for the expression and purification of a stable form of human adenosine A2aR. WT and C-terminally truncated adenosine A2aR constructs were expressed in the methylotrophic yeast Pichia pastoris. Very high-level expression (11 mg/L, 222 pmol/mg) was obtained for an adenosine A2aR truncated at residue V334. The functional yield following both solubilisation and purification was monitored by radioligand binding analysis using [3H] ZM241385 as a means of optimising recovery of receptor. Solubilisation trials revealed that enrichment of functional recovery of V334 adenosine A2aR was only possible in the presence of cholesterol. A two-step purification protocol including Flag tag affinity chromatography followed by TEV cleavage and reverse His-trap resulted in a highly homogenous and pure sample with a specific activity of 21 nmol/mg, close to the theoretical maximum of 24 nmol/mg. The purified receptor is functionally stable and degradation resistant for a period of 15 days at 4°C and 20°C. G-protein peptides were generated as potential tools for cocrystallisation. The protein was submitted to crystallisation trials in the presence and absence of the peptides using the lipidic cubic phase technique.
Supervisor: Byrne, Bernadette ; Iwata, So Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral