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Cellular and Molecular Neurobiology

, Volume 11, Issue 1, pp 143–156 | Cite as

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme

  • Baruch Velan
  • Chanoch Kronman
  • Haim Grosfeld
  • Moshe Leitner
  • Yehoshua Gozes
  • Yehuda Flashner
  • Tamar Sery
  • Sara Cohen
  • Revital Ben-Aziz
  • Shlomo Seidman
  • Avigdor Shafferman
  • Hermona Soreq
Article

Summary

  1. 1.

    Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase.

     
  2. 2.

    The recombinant gene product exhibits biochemical traits similar to native “true” acetylcholinesterase as manifested by characteristic substrate inhibition, aK m of 117µM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51.

     
  3. 3.

    The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization.

     
  4. 4.

    The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms.

     
  5. 5.

    In conclusion, the catalytic subunit expressed from the hydrophylic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

     

Key words

acetylcholinesterase transient transfection 293 cells cytomegalovirus (CMV) promoter 

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Copyright information

© Plenum Publishing Corporation 1991

Authors and Affiliations

  • Baruch Velan
    • 1
  • Chanoch Kronman
    • 1
  • Haim Grosfeld
    • 1
  • Moshe Leitner
    • 1
  • Yehoshua Gozes
    • 1
  • Yehuda Flashner
    • 1
  • Tamar Sery
    • 1
  • Sara Cohen
    • 1
  • Revital Ben-Aziz
    • 2
  • Shlomo Seidman
    • 2
  • Avigdor Shafferman
    • 1
  • Hermona Soreq
    • 2
  1. 1.Department of BiochemistryIsrael Institute for Biological ResearchNess-ZionaIsrael
  2. 2.Department of Biological Chemistry, The Life Sciences InstitueThe Hebrew UniversityJerusalemIsrael

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