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Received: 2020-09-04

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Liujia SHI


Shoufeng WANG


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Journal of Zhejiang University SCIENCE B 2021 Vol.22 No.3 P.204-213


Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides

Author(s):  Liujia SHI, Fangfang YANG, Yanyan XU, Shoufeng WANG

Affiliation(s):  Institute of Pharmaceutical Biotechnology, School of Medicine, Zhejiang University, Hangzhou 310058, China; more

Corresponding email(s):   sfwang@zju.edu.cn

Key Words:  Acetylcholinesterase (AChE), Pichia pastoris, Enzyme activity determination, Pesticide sensitivity

Liujia SHI, Fangfang YANG, Yanyan XU, Shoufeng WANG. Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides[J]. Journal of Zhejiang University Science B, 2021, 22(3): 204-213.

@article{title="Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides",
author="Liujia SHI, Fangfang YANG, Yanyan XU, Shoufeng WANG",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides
%A Liujia SHI
%A Fangfang YANG
%A Yanyan XU
%A Shoufeng WANG
%J Journal of Zhejiang University SCIENCE B
%V 22
%N 3
%P 204-213
%@ 1673-1581
%D 2021
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2000525

T1 - Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides
A1 - Liujia SHI
A1 - Fangfang YANG
A1 - Yanyan XU
A1 - Shoufeng WANG
J0 - Journal of Zhejiang University Science B
VL - 22
IS - 3
SP - 204
EP - 213
%@ 1673-1581
Y1 - 2021
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B2000525

acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris. The pPIC9K-Drosophila melanogaster acetylcholinesterase (DmAChE)-like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL⋅min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.


结论:在本研究中,我们构建了基因工程的GS115-pPIC9K-DmAChE-like菌株,并在实验室水平诱导了基因表达。我们测试了各个发酵上清液的酶活性,证明了DmAChE-like具有乙酰胆碱酯酶活性。通过Tricine-SDS-PAGE,我们发现特异的蛋白条带为4.3 kDa,与预期结果一致。农药敏感性试验发现DmAChE-like对大多数选择的有机磷农药敏感,表明它可以用作有机磷农药检测酶抑制方法的新酶源。


Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


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