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Journal of Integrative Agriculture  2017, Vol. 16 Issue (06): 1358-1366    DOI: 10.1016/S2095-3119(16)61508-3
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High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application
LIU Niu1, CHE Jun1, 2, LAI Duo1, 3, WEN Jin-jun1, XU Han-hong1

1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources & Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education/South China Agricultural University, Guangzhou 510642, P.R.China

2 Shenzhen Longgang Inspection Station of Non-pollution Agricultural Products, Shenzhen 510172, P.R.China

3 Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China

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Abstract  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 ?rst precipitated with ammonium sulfate (50% saturation) and then puri?ed with procainamide af?nity column chromatography.  The enzyme was puri?ed 12.1-fold with a yield of 22.8% and a high speci?c 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.
Keywords:  Plutella xylostella      acetylcholinesterase      Pichia pastoris,      high-level expression  
Received: 16 June 2016   Accepted:
Fund: 

This work was supported by a grant from the Public Benefit Research Foundation of China (200903052) and the Science and Technology Department of Guangdong Province, China (2009A020101003).

Corresponding Authors:  XU Han-hong, Tel: +86-20-85285127, E-mail: hhxu@scau.edu.cn    

Cite this article: 

LIU Niu, CHE Jun, LAI Duo, WEN Jin-jun, XU Han-hong . 2017. High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application. Journal of Integrative Agriculture, 16(06): 1358-1366.

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