Figure 1.
The chemical structures of the different compounds tested.
(A) structure of the natural inhibitor of AChE, (B) structures of the CB pesticides, (C) structures of the OP pesticides.
Table 1.
Primers used for cloning bmace and AGα1 genes and generating the synthetic gene encoding bmace-AGα1 fusion protein.
Figure 2.
Construction of the BmAChE-display with the P. pastoris expression system based on α-agglutinin.
The bmace and AGα1 DNA segments were spliced using overlap extension PCR to assemble the bmace-AGα1 gene and inserted into pPIC9K for the pPIC9K-bmace-AGα1 construction.
Figure 3.
Fluorescence microscopy assay of recombinant P. pastoris cells displaying BmAChE: The fluorescence at 519 nm emitted with excitation at 495 nm was observed by fluorescence microscopy.
(a) and (c), phase micrographs of recombinant yeast cells; (b) and (d), fluorescent micrographs of recombinant yeast cells. GS115/pPIC9K-bmace-AGα1 (a, b); GS115 as a control (c, d).
Figure 4.
Flow cytometry detection of BmAChE displayed on the recombinant yeast surface.
Figure 5.
P. pastoris display of BmAChE on an MM-B plate: 1, GS115; 2, GS115/pPIC9K-bmace-AGα1; 3–10, GS115/pPIC9K-bmace-AGα1 inhibited by different concentrations of eserine (clone 3: 10−2 M, clone 4: 10−3 M, clone 5: 10−4 M, clone 6: 10−5 M, clone 7: 10−6 M, clone 8: 10−7 M, clone 9: 10−8 M, clone 10: 10−9 M).
Figure 6.
Inhibition curve of displayed BmAChE.
(A), inhibition curve of displayed BmAChE for eserine; (B), inhibition curve of displayed BmAChE for CB pesticide (n = 3); and (C), inhibition curve of displayed BmAChE for OP pesticides (n = 3). B, the average absorbance at the indicated concentrations; B0, the average absorbance at zero concentration. The data were fitted with a four-parameter-logistic equation to calculate the IC50 using OriginPro 7.5 software. The data points are mean values and the errors observed from triplicate determinations.
Table 2.
The median inhibition concentration (IC50) of displayed BmAChE for five CB and seven OP pesticides.