Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin
| Autor: | Marchot, P., Ravelli, R. B. G., Raves, M. L., Bourne, Y., Vellom, D. C., Kanter, J., Camp, S., Joel Sussman, Taylor, P. |
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| Přispěvatelé: | University of California [San Diego] (UC San Diego), University of California (UC), Centre National de la Recherche Scientifique (CNRS), Bijvoet Center for Biomolecular Research [Utrecht], Utrecht University [Utrecht], Brookhaven National Laboratory [Upton, NY] (BNL), UT-Battelle, LLC-Stony Brook University [SUNY] (SBU), State University of New York (SUNY)-State University of New York (SUNY)-U.S. Department of Energy [Washington] (DOE), Weizmann Institute of Science [Rehovot, Israël], The Scripps Research Institute [La Jolla, San Diego], European Synchrotron Radiation Facility (ESRF), U.S. Department of Energy [Washington] (DOE)-UT-Battelle, LLC-Stony Brook University [SUNY] (SBU), State University of New York (SUNY)-State University of New York (SUNY), University of California |
| Předmět: |
Elapid Venoms
DNA Complementary crystallization Base Sequence Protein Conformation [SDV]Life Sciences [q-bio] [SDV.BA]Life Sciences [q-bio]/Animal biology Molecular Sequence Data peptide-macromolecule complex Crystallography X-Ray snake toxin Cell Line Mice Acetylcholinesterase Centrifugation Density Gradient Animals fasciculin Electrophoresis Polyacrylamide Gel Amino Acid Sequence Cholinesterase Inhibitors Research Article |
| Zdroj: | Scopus-Elsevier Protein Science Protein Science, 1996, 5 (4), pp.672-679. ⟨10.1002/pro.5560050411⟩ Protein Science, Wiley, 1996, 5 (4), pp.672-679. ⟨10.1002/pro.5560050411⟩ |
| ISSN: | 0961-8368 1469-896X |
| DOI: | 10.1002/pro.5560050411⟩ |
| Popis: | International audience; 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. |
| Databáze: | OpenAIRE |
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