Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris

PLoS One. 2013 Aug 5;8(8):e70451. doi: 10.1371/journal.pone.0070451. Print 2013.

Abstract

A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10(-8) M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholinesterase / genetics
  • Acetylcholinesterase / metabolism*
  • Animals
  • Bombyx / enzymology*
  • Carbamates / pharmacology
  • Enzyme Activation / drug effects
  • Organophosphorus Compounds / pharmacology
  • Physostigmine / pharmacology
  • Pichia / enzymology*
  • Pichia / genetics
  • Pichia / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Carbamates
  • Organophosphorus Compounds
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Physostigmine
  • Acetylcholinesterase

Grants and funding

This research was granted by the National Natural Science Foundation of China (31201361, 31271866), the National Science & Technology Pillar Program of China (2012BAD31B0302), Science and Technology Plan Projects in Guangdong Province (2012A020100002, 2010A032000001-4) and the Doctoral Innovation Program of Hopson Zhujiang Education Fund (H2011001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.