Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris.
A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10(-8) M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.
Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou, Guangdong Province, China.
Jie-Xian
JX
Dong
Xi
X
Xie
Yong-Sheng
YS
He
Ross C
RC
Beier
Yuan-Ming
YM
Sun
Zhen-Lin
ZL
Xu
Wei-Jian
WJ
Wu
Yu-Dong
YD
Shen
Zhi-Li
ZL
Xiao
Li-Na
LN
Lai
Hong
H
Wang
Jin-Yi
JY
Yang
D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D002219:Carbamates / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010830:Physostigmine / Q000494:pharmacology; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D002219:Carbamates; D009943:Organophosphorus Compounds; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D010830:Physostigmine; D000110:Acetylcholinesterase