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Position: Home > Articles > High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application Journal of Integrative Agriculture 2017,16 (6)

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

作者：

Liu Niu;Che Jun;Lai Duo;Wen Jin-jun;Xu Han-hong

单位：

South China Agr Univ, Minist Educ, State Key Lab Conservat & Utilizat Subtrop Agrobi, Guangzhou 510642, Guangdong, Peoples R China;Shenzhen Longgang Inspect Stn Nonpollut Agr Prod, Shenzhen 510172, Peoples R China;South China Agr Univ, Minist Educ, Key Lab Nat Pesticide & Chem Biol, Guangzhou 510642, Guangdong, Peoples R China;Guangdong Acad Agr Sci, Inst Fruit Tree Res, Guangzhou 510640, Guangdong, Peoples R China

关键词：

Plutella xylostella;acetylcholinesterase;Pichia pastoris;high-level expression

摘要：

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

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