Our first goal should be to bring 20-30 receptors to the crystallisation stage, starting from the 7 well purified receptors and in the same time purifying or refolding the remaining 30 well expressed/characterised targets from Pichia, SFV and E. coli. These crystallisation trials will be limited to 1000 – 2000 conditions and help identify priority targets.. This initial goal should be accomplished within the first year with the major delivery of initial diffracting GPCRs crystals.
As our second goal, we will focus on few promising targets, around 10 out of the initial 20-30 GPCRs from the first step. Selection of priority targets will be based on initial crystallisation success or promising stability assessments demonstrated by various biophysical characterisations. Starting from larger batches, we will focus on optimisation of the GPCR crystals obtained in the first part of the initiative. Intensive crystallisation screens and high throughput stabilisation will be performed using alternative detergents and additives as well as exploiting the interaction between GPCRs and other proteins such as G-proteins and arrestins.
Aim one: To obtain initial diffracting crystals from the screening of 20 to 30 purified GPCRs.
Aim Two: To obtain several GPCR structures by intensive crystallisation optimisation and high throughput stabilisation screening approaches.
Development of alternative affinity based purification approaches
We will also explore the possibility of developing alternative purification methods based on ligand or arresting affinity based columns using biotinylated ligands, tagged arrestin or arrestin fusion proteins.