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

Florian Nachon, Yvain Nicolet, Nathalie Viguié, Patrick Masson, Juan C. Fontecilla-Camps, Oksana Lockridge

Research output: Contribution to journalArticlepeer-review

103 Scopus citations

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 Å resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 Å, c = 124.9 Å, giving a Vm of 2.73 Å3 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.

Original languageEnglish (US)
Pages (from-to)630-637
Number of pages8
JournalEuropean Journal of Biochemistry
Volume269
Issue number2
DOIs
StatePublished - 2002

Keywords

  • Butyrylcholinesterase
  • Crystallization
  • N-glycosylation
  • Site-directed mutagenesis
  • X-ray diffraction

ASJC Scopus subject areas

  • Biochemistry

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