Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin
Résumé
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, Fasl-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm × 0.5 mm × 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 P6122 or P6522 with unit cell dimensions a = b = 75.5 Å, c = 556 Å, giving a Vm value of 3.1 Å3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fasl-mAChE crystallizes in the same space group with identical cell dimensions.