Protein Science, Vol 5, Issue 4 672-679, Copyright © 1996 by Cold Spring Harbor Laboratory Press

ARTICLE

Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin

P. MARCHOT, RBG. RAVELLI, M. L. RAVES, Y. BOURNE, D. C. VELLOM, J. KANTER, S. CAMP, J. L. SUSSMAN and P. TAYLOR
Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0636 Permanent address: Laboratoire de Biochimie, CNRS, Unite de Recherche Associee 1455, Institut Federatif de Recherche Jean Roche, Universite d'Aix-Marseille II, Faculte de Medecine Secteur Nord, 13916 Marseille cedex 20, France.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm X 0.5 mm X 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a V(m) value of 3.1 A(3)/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.
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