Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.
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.
Marchot
P
P
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Ravelli
R B
RB
Raves
M L
ML
Bourne
Y
Y
Vellom
D C
DC
Kanter
J
J
Camp
S
S
Sussman
J L
JL
Taylor
P
P
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002800:Cholinesterase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D004546:Elapid Venoms / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation
D002800:Cholinesterase Inhibitors; D018076:DNA, Complementary; D004546:Elapid Venoms; C044937:fasciculin; D000110:Acetylcholinesterase