关键词：乙酰胆碱酯酶；基因；单体；神经毒剂
摘 要：The human acetylcholinesterase (hAChE) gene was cloned into the pHLsec expression vector. The recombinant enzyme (rhAChEmD) was expressed on a large scale in adherent 293T cells. It was secreted as a monomeric species, purified by affinity chromatography, and deglycosylated with PNGase F. A crystallization screen, using the Mosquito crystallization robot, identified conditions for formation of diffraction-quality crystals, in 0.025% dichloromethane/12% PEG 20,000/0.1M imidazole, pH7.0. The crystals formed in the hexagonal space group P3112, with cell constants a=125.31, b=125.31, c=131.40 , and one monomer per asymmetric unit. A complete dataset, to 2.9 resolution, was collected at 100 K at the ESRF (Grenoble, France). The structure was solved by molecular replacement, resulting in an Rfree of 23.74%, and Rwork of 19.31%, for all data to 2.9 . The coordinates and structures factors have been deposited in the PDB, with IDcode 4PQE. As an initial step for synthesis of a GF surrogate, the chloridate, CH3P(O)(O-cyclohexyl)Cl, has been prepared, and will be reacted with coumarin. Conditions have also been developed for expression of a full-length rhAChE construct, rhAChET that assembles to form the physiological tetramer.