Intracellular domain of nicotinic acetylcholine receptor: Expression for structural investigations

On the structural map of the nicotinic acetylcholine receptor (nAChR) the intracellular domain remains "a blank space". In order to investigate the structure and function of this intracellular domain we are expressing the intracellular loop of the nAChR delta-subunit individually and the loops of all subunits (alpha(2)betagammadelta) collectively, using a polycistronical vector. E.coli cells express the individual loop in insoluble form, after refolding and purification a protein concentration of 0.3 mg/ml can be obtained. The CD spectra of the fusion protein show a high percentage of ordered structure. The coexpression of the loops in the same cell did not improve protein solubility, after refolding the protein yield remains low (similar to0.05 mg/ml). By cross-linking experiments the formation of distinct oligomers has been demonstrated.