Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization.

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Nachon F, Nicolet Y, Viguie N, Masson P, Fontecilla-Camps JC, Lockridge O

Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization.

Eur J Biochem. 2002 Jan;269(2):630-7.

PubMed ID
11856322 [ View in PubMed
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Abstract

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.

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