Journal of Integrative Agriculture (Jun 2017)

High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

  • Niu LIU,
  • Jun CHE,
  • Duo LAI,
  • Jin-jun WEN,
  • Han-hong XU

Journal volume & issue
Vol. 16, no. 6
pp. 1358 – 1366

Abstract

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The acetylcholinesterase 2 (AChE2) cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115. One transformant with high-level expression of the recombinant AChE (rAChE, 23.2 U mL−1 in supernatant) was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate. The maximum production of rAChE reached about 11.8 mg of the enzyme protein per liter of culture. The rAChE was first precipitated with ammonium sulfate (50% saturation) and then purified with procainamide affinity column chromatography. The enzyme was purified 12.1-fold with a yield of 22.8% and a high specific activity of 448.3 U mg−1. It was sensitive to inhibition by methamidophos and pirimicarb, the calculated 50% inhibitory concentration (IC50) values of the two pesticides were 0.357 and 0.888 mg L−1, respectively, and the calculated 70% inhibitory concentration (IC70) values were 0.521 and 0.839 mg L−1, respectively. The results suggested that it has a potential application in the detection of pesticide residues.

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