Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization.
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.
Nachon
Florian
F
Centre de Recherches du Service de Santé des Armées, Unité d'Enzymologie, La Tronche, France. florian@nachon.net
Nicolet
Yvain
Y
Viguié
Nathalie
N
Masson
Patrick
P
Fontecilla-Camps
Juan C
JC
Lockridge
Oksana
O
D000595:Amino Acid Sequence; D000818:Animals; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D002091:Butyrylcholinesterase