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

文献类型： 外文期刊

第一作者： Liu Niu

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

作者机构：

关键词： Plutella xylostella;acetylcholinesterase;Pichia pastoris;high-level expression

期刊名称：JOURNAL OF INTEGRATIVE AGRICULTURE （ 影响因子：2.848； 五年影响因子：2.979 ）

ISSN： 2095-3119

年卷期： 2017 年 16 卷 6 期

页码：

收录情况： SCI

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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