Marchot_1996_Prot.Sci_5_672

Reference

Title : Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin - Marchot_1996_Prot.Sci_5_672
Author(s) : Marchot P , Ravelli RB , Raves ML , Bourne Y , Vellom DC , Kanter J , Camp S , Sussman JL , Taylor P
Ref : Protein Science , 5 :672 , 1996
Abstract :

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 Vm value of 3.1 A3/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.

PubMedSearch : Marchot_1996_Prot.Sci_5_672
PubMedID: 8845756
Gene_locus related to this paper: mouse-ACHE

Related information

Inhibitor Fasciculin2
Gene_locus Fasciculin2    mouse-ACHE

Citations formats

Marchot P, Ravelli RB, Raves ML, Bourne Y, Vellom DC, Kanter J, Camp S, Sussman JL, Taylor P (1996)
Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin
Protein Science 5 :672

Marchot P, Ravelli RB, Raves ML, Bourne Y, Vellom DC, Kanter J, Camp S, Sussman JL, Taylor P (1996)
Protein Science 5 :672