The objective of our research is the resolution of a number of GPCR structures within 2008. The following major actions are planned in order to achieve this objective.
Seven successfully purified receptors are subject to immediate large scale production and crystallisation trials. In parallel, solubilisation and purification screening of 30 further well-expressed targets (over 10 pmol/mg in Pichia and SFV) will be performed within one year. If necessary, further targets will be added to the list. Any receptor, which can be purified at levels of more than 1 mg, will be immediately subjected to crystallisation. The receptors which have strong affinity ligands and/or show high activity after solubilisation/purification will be given higher priority within the list. Priority targets which are well expressed in E. coli as inclusion bodies are subject to refolding attempts.
- Initial crystallisation trials on 20-30 targets
Several milligrams of target GPCRs have been purified in the presence of strong affinity ligands. The rational crystallisation screening strategies developed at Imperial College (22% success rate for 36 purified bacterial membrane proteins) will be applied to these receptors using automated nanoliter crystallisation technology. Receptors will always be crystallised in the presence of strong affinity ligands in order to improve stability. Systematic co-crystallisation with various ligands, G-proteins and arrestins are also performed. The obtained crystals will be subjected to high throughput screening at a synchrotron radiation facility and optimised using sets of additives and secondary detergents.
- Large scale production and intensive crystallisation of a limited number of promising targets
Since stability is the key for successful crystallisation of GPCRs, a few receptors, for which high throughput quality control is available, are used as model systems to study stable solubilisation and purification of GPCRs. Optimised stabilisation conditions could then be applied to all the targets at the crystallisation stage.
- Stabilisation studies on a limited number of GPCRs for crystallisation
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