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Báo cáo khoa học: Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase

Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophos-phorus pesticides andnerveagents.The relative contribution of each N-linked glycan for the solubility, the stability and the secretionof the enzymewas investigated. Arecombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. | Eur. J. Biochem. 269 630-637 2002 FEBS 2002 Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase Expression purification characterization and crystallization Florian Nachon1 Yvain Nicolet2 Nathalie Viguie1 Patrick Masson1 Juan C. Fontecilla-Camps2 and Oksana Lockridge3 1 Centre de Recherches du Service de Sante des Armees Unite d Enzymologie La Tronche France 2Laboratoire de Cristallographie et Cristallogenese des Proteines Institut de biologie structurale J.P. Ebel Grenoble France 3 University of Nebraska Medical Center Eppley Research Institute Omaha NE USA Human butyrylcholinesterase BChE EC 3.1.1.8 is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained they were improved by recrystallization and found to diffract to 2.0 A resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a b 154.7 A c 124.9 A giving a Vm of 2.73 A3 per Da estimated 60 solvent for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general. Keywords butyrylcholinesterase crystallization N-glycosy-lation site-directed mutagenesis X-ray diffraction. Acetylcholinesterase AChE EC 3.1.1.7 and butyrylcholinesterase BChE EC 3.1.1.8 are closely related serine hydrolases with different substrate specificity .