Engineering of a monomeric and lowâglycosylated form of human butyrylcholinesterase: Expression, purification, characterization and crystallization
2002
Nachon, Florian | Nicolet, Yvain | Viguié, Nathalie | Masson, Patrick | FontecillaâCamps, Juan C. | Lockridge, Oksana
Human butyrylcholinesterase (BChE; ECâ3.1.1.8) 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 Nâlinked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine Nâglycosylation sites and the Câterminal 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 Xâray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0âà resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions aâ=âbâ=â154.7âà , câ=â124.9âà , giving a Vm of 2.73âà 3 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.
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