Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin
| Autor: | Joan R. Kanter, Joel L. Sussman, Palmer Taylor, Pascale Marchot, Raimond B. G. Ravelli, Shelley Camp, Mia L. Raves, Daniel C. Vellom, Yves Bourne |
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| Rok vydání: | 1996 |
| Předmět: |
chemistry.chemical_classification
0303 health sciences Stereochemistry 030302 biochemistry & molecular biology HEK 293 cells Biology Biochemistry law.invention 03 medical and health sciences chemistry.chemical_compound Monomer Enzyme Protein structure chemistry Affinity chromatography law Complementary DNA Crystallization Molecular Biology Peptide sequence 030304 developmental biology |
| Zdroj: | Protein Science. 5:672-679 |
| ISSN: | 0961-8368 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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