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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.


Introduction
The intensive use of carbamate (CB) and organophosphorus (OP) pesticides in recent years has led to potentially dangerous effects on human and animal health. The control of pesticide residues in food and the environment is of great importance to minimize the risk to consumers and environmental animal species. Routinely, CB and OP pesticide residues are measured by instrumental methods, such as gas chromatography, liquid chromatography and gas chromatography-tandem mass spectrometry [1,2,3]. There is a growing interest in more rapid and low-cost field-portable detection systems. A promising approach involves the use of screening enzyme-linked immunoassays [4]. However, these assays require broad-specificity antibodies that are difficult to develop. Nevertheless, an enzyme-based method was demonstrated to be an efficient and rapid method for the detection of pesticides because it was inexpensive, allowed high sample throughput, and was easily adapted for use in Asian markets [5].
Previously, acetylcholinesterase (AChE), aldehyde dehydrogenase, alkaline and acid phosphatase, butyrylcholinesterase, organophosphorus hydrolase and tyrosinase have been investigated for their ability to detect pesticides in water and other matrices such as soil, food and beverages [6]. However, AChE has been most often used for enzymatic detection of pesticides because of its broad substrate specificity and good sensitivity [6].
AChE is a key enzyme in the cholinergic system that regulates the level of acetylcholine and terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine in the synaptic cleft [7,8]. The enzyme activity of AChE can be inhibited by CB and OP pesticides. Therefore, it is feasible to use AChE for the detection of CB and OP pesticides based on the degree of AChE activity inhibition [9]. AChE has been isolated by traditional extraction methods from natural tissues [10,11,12] or from secretions of engineered cells [8,13]. Isolation from these areas requires an enzyme purification step, which leads to higher preparation costs. However, natively displayed molecules on the surface of cells presents another option, which is currently of great interest. Many heterologous proteins and polypeptides have been displayed on the surface of cells, and these displays have been widely used [14,15,16,17,18]. The use of displayed molecules on the cell surface can save tedious purification steps required for enzymes used in traditional immobilization methods. Further, protein engineering can help generate a surface display of enzymes that can be used in efficient high-throughput screening methods for residue detection. In cell surface display development, the anchor protein is a necessary component. The most frequently used anchor is the N-terminal fusion display of a-agglutinin from Saccharomyces cerevisiae (S. cerevisiae), which is composed of a secretion-signal region, an active region, a serine-and threonine-rich support region, and a putative glycosylphosphatidylinositol anchor-attachment protein. AGa1 protein is one of the aagglutinins in N-terminal fusion displays [19]. Different enzymes used for the detection of pesticides, like organophosphorus hydrolase [20] and mouse AChE [21], have been expressed on the surface of microorganisms. In this study we took advantage of the domestic silkworm, Bombyx mori as the ace gene source to investigate the sensitivity of the displayed AChE for pesticides. Domesticated silkworms have not suffered from pesticide selection, severe competition for food, or from finding good mating partners over the last number of decades. As a result, the sensitivity of AChE in domestic silkworms would be expected to be preserved and be more sensitive than the enzyme from the wild-type source [22], suggesting that AChE from Bombyx mori may be a remarkable reagent for pesticide detection.
This study was conducted with the aim to construct a cell surface display system for Bombyx mori AChE (BmAChE). The work may lay the foundation for further sensitivity improvement by developing a displayed AChE system through recombinant molecular methods and application of whole-cell biosensors for the broad-specificity detection of CB and OP pesticides. Here we displayed the BmAChE on Pichia pastoris (P. pastoris) for the first time. We cloned the AChE gene from Bombyx mori and the anchor protein gene AGa1 from Saccharomyces cerevisiae (S. cerevisiae). We then constructed a stable P. pastoris cell surface display system for the recombinant BmAChE. The display of the recombinant enzyme on the surface of the yeast was then used for the detection of CB and OP pesticides, which resulted in the development of a rapid, easy and sensitive analytical method useful for the detection of pesticide residues.

Strains and Media
Escherichia coli (E. coli) DH5a stored in our laboratory was used as the host for recombinant DNA manipulation. The P. pastoris GS115 strains and the integrative expression vector (pPIC9K) were obtained from Invitrogen Biotechnology Co. (Shanghai, China).

Cloning and Assembly of the bmace-AGa1 Gene
The construction scheme for the plasmid containing the bmace-AGa1 fusion gene is shown in Fig. 1; DNA fragments encoding for BmAChE were amplified with the constructed vector pPIC9Kbmace [23] as a template without the signal peptides and the hydrophobic amino acid tail gene. The PCR process was performed using PrimerSTAR DNA Polymerase and the amplification experiment was run at a melting temperature of 94uC for 1 min, annealing at 58uC for 1 min, and extension at 72uC for 2 min, with a 30 cycle repeat. Primers used for PCR amplification containing the FLAG tag at 59 and partial linker at 39 were the two oligonucleotides F1 and R1, respectively ( Table 1). The genome of S. cerevisiae was extracted using the yeast genome extraction kit, and the AGa1 gene was amplified using the genome as template and the F2 and R2 primers listed in Table 1. The purified bmace and AGa1 DNA segments (50 ng each) were spliced using overlap extension PCR to assemble the bmace-AGa1 gene with the (Gly 4 Ser) 3 linker. Then, the bmace-AGa1 gene was amplified using the F1 and R2 primers ( Table 1). The PCR amplification products were purified by an agarose gel DNA purification kit and stored at 220uC.

Construction of the Plasmid for Cell Surface Display
The resulting PCR products from the above step were digested with the restriction enzymes Mlu I and Not I, and then ligated into the expression vector pPIC9K. The ligated products were transformed into competent E. coli DH5a cells for propagation of the recombinant plasmid. The recombinant plasmid pPIC9Kbmace-AGa1 was confirmed by restriction enzyme digestion and DNA sequencing.

P. pastoris Transformation and Selection
Linearized vectors were transformed into P. pastoris as previously described [24]. Transformed cells were spread on MD plates and incubated at 30uC for 3 d to select His + transformants. Genomic integration was confirmed by performing PCR on genomic DNA with the AOX-F and AOX-R primers ( Table 1).

Expression of the bmace-AGa1 Gene
The recombinant P. pastoris clone was grown in 20 mL BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol and 100 mM potassium phosphate (pH 6.0)) in shake culture at 30uC for 24 h until the OD 600 reached a value of more than 4. The culture (5 mL) was centrifuged at 30006g for 5 min. The cells were induced by re-suspension with 20 mL BMMY medium (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 0.5% methanol and 100 mM potassium phosphate (pH 6.0)) and the resulting OD 600 was approximately 1. The induction was continued at 28uC for 4 more d by adding 200 mL of 100% methanol to the cultures daily.

Characterization of the Displayed BmAChE by Fluorescence Microscopy
The immunofluorescent labeling of yeast cells was carried out as follows: A cell suspension was centrifuged at 80006g for 1 min, and the collected cells were washed three times with 0.01 M phosphate buffered saline (PBS) (8.0 g/L NaCl, 1.44 g/L Na 2 HPO 4 N12H 2 O, 0.2 g/L KCl, 0.24 g/L KH 2 PO 4 (pH 7.4)). The cells were suspended and blocked with PBS containing 1% bovine serum albumin (BSA) for 0.5 h (OD 600 = 1.0). Anti-FLAG IgG (1 mL) was added to the 200 mL cell suspension and incubated at room temperature for 1.

Flow Cytometry Detection
The number of yeast cells displaying BmAChE was determined using a flow cytometer (BD FAcscalibur, CA, USA) with a 488 nm excitation wavelength and a 525 nm emission wavelength to estimate the percentage of BmAChE molecules displayed.

Enzyme Activity Determination of the Displayed BmAChE
The activity of the displayed BmAChE was evaluated spectrophotometrically at 405 nm according to Ellman et al [25]. using the substrate ATC and the chromogenic reagent DTNB. A cell suspension (100 mL) of transformed P. pastoris was centrifuged at 80006g for 2 min and the cells were weighed. The collected cells (approximately 1.03610 7 cells as determined by a hemocytometer) were washed three times with potassium phosphate buffer (3.075 mL of a 1 M K 2 HPO 4 solution combined with 1.925 mL of a 1 M KH 2 PO 4 solution (pH 7.0)) and re-suspended in 780 mL potassium phosphate buffer (pH 7.0). The enzymatic reaction was activated by consecutively adding 100 mL of 1 mM ATC and 7.8 mM DTNB. The reaction mixture was incubated at room temperature for 5 min and stopped with 20 mL of 1610 27 M eserine. After centrifugation of the reaction mixture, the supernatant was used to measure the OD at 405 nm with an ELISA reader (Multiscan MK3, Labsystem Co., Finland). One unit of AChE activity was defined as the amount of enzyme hydrolyzing 1 mmol of ATC in 1 min with 1 g of wet cells.  Table 1. Primers used for cloning bmace and AGa1 genes and generating the synthetic gene encoding bmace-AGa1 fusion protein.

Inhibition of Displayed BmAChE
Inhibition of the displayed BmAChE was carried out in the presence of eserine [26], a well-known AChE inhibitor previously employed to study the enzyme. Transformants were inoculated on MM-B agar plates (1.34% YNB (v/v), 0.00004% biotin, 1% methanol, 100 mM potassium phosphate buffer (pH 7.0) and 1.8% agarose) at 28uC for 3 d. Then 10 mL of 50 mM potassium phosphate buffer (pH 7.0) was placed on one GS115 colony (negative control) and on one GS115/pPIC9K-bmace-AGa1 colony (positive control), and 5 mL of 50 mM potassium phosphate buffer (pH 7.0) and 5 mL of different concentrations of eserine (10 22 -10 29 M) were placed on 8 other positive colonies. Following a 10 min reaction period, 3 mL of 10 mM ATC and 3 mL of 7.8 mM DTNB were added to each colony, and the colony color was observed after 10 min at 37uC. Also, an inhibition study of the displayed BmAChE was performed according to Ellman's method [25] using the yeast cell suspension and different concentrations of eserine.

Detection of CB and OP Pesticides Using the Displayed BmAChE
Five CB pesticides (carbofuran, carbosulfan, isoprocarb, methiocarb and methomyl) and seven OP pesticides (dichlorphos, dimethoate, isocarbophos, malathion, methamidophos, parathion and trichlorphon) were tested ( Figure 1). The induced cell suspension was centrifuged at 80006g for 2 min, re-suspended and adjusted to an OD 600 of 2 using 50 mM potassium phosphate buffer (pH 7.0). A volume of 120 mL transformed P. pastoris cell suspension (approximately 0.25610 7 ) was mixed with the same volume but different concentrations of CB and OP pesticides. After a 5 min incubation at room temperature, 30 mL of 10 mM ATC and 7.8 mM DTNB were consecutively added, and 20 mL of 1610 27 M eserine was added to stop the reaction. The reaction mixture was centrifuged at 80006g for 2 min and the suspension was removed to microlon plates. The activity of AChE was measured using the multilabel counter at 405 nm. The median inhibition concentration (IC 50 ) for each compound was calculated based on the Log-dose versus probit regression [27]. The lowest concentration that could be detected was measured according to the Inhibition Rate (B/B 0 ).

Results and Discussion
Construction of the BmAChE Yeast Surface Display System Using P. pastoris The plasmid for surface display of BmAChE was constructed as shown in Fig. 2. The amplification of bmace generated an approximate 1900 bp DNA fragment, while the AGa1 gene generated an expected 1000 bp fragment. PCR amplification of the assembled bmace-AGa1 gene produced an expected 2900 bp fragment ( Figure S1, see supplementary data for the nucleic acid sequence in the Supporting Information). The bmace-AGa1 gene with a FLAG tag (eight amino acids) at the N-terminus of AChE was subcloned into the expression vector pPIC9K. The results from sequencing indicated the recombinant plasmid pPIC9Kbmace-AGa1 had been successfully constructed. PCR amplification using AOX-F and AOX-R primers (Table 1) with the genome of the selected transformants as template produced the 2900 bp amplified fragment, indicating that the constructed vectors were integrated into the genome of P. pastoris GS115.

Characterization of the Displayed BmAChE by Fluorescence Microscopy and Flow Cytometry
The display of BmAChE on the yeast cell surface was evaluated by immunofluorescence microscopy. Fluorescence was observed on the cell surface of the pPIC9K-bmace-AGa1 transformant strains Figure 2. Construction of the BmAChE-display with the P. pastoris expression system based on a-agglutinin. The bmace and AGa1 DNA segments were spliced using overlap extension PCR to assemble the bmace-AGa1 gene and inserted into pPIC9K for the pPIC9K-bmace-AGa1 construction. doi:10.1371/journal.pone.0070451.g002 using a fluorescence microscope, and fluorescence was not observed from the control cells (Fig. 3). The images demonstrated that the bmace-AGa1 fusion protein was anchored on the P. pastoris surface.
The expression of the BmAChE fusion protein on the surface of P. pastoris was further analyzed by indirect immunofluorescence labeling using flow cytometry (Fig. 4). A difference was detected in the amounts of BmAChE-a-agglutinin fusion protein expression obtained from P. pastoris pPIC9K-bmace-AGa1 transformants. Fluorescence was detected in about 25% of the constructed cells. These studies confirmed that BmAChE was displayed on the cell surface of P. pastoris.

Enzyme Activity Determination Using Displayed BmAChE
AChE activity was measured based on the Ellman method [25] using ATC and DTNB. The hydrolytic activity of the BmAChE enzymes displayed on the surface of the cells was 787.7 U/g (wet cell weight) after being induced with methanol for 4 days at 28uC.

The inhibition analysis of displayed BmAChE
The inhibition characteristics of eserine, the AChE specific inhibitor, were performed on MM-B plates. As shown in Fig. 5, the color of the original P. pastoris GS115 colonies was white (Fig. 5, colony 1), while the color of the transformed P. pastoris GS115/ pPIC9K-bmace-AGa1 colonies was yellow (Fig. 5, colony 2). When the concentration of eserine was between 10 27 -10 29 M, the color of the pPIC9K-bmace-AGa1 colonies gradually turned yellow (Fig. 5, colony 8-10). When the concentration of eserine was 10 29 M (Fig. 5, colony 10), the color was close to the positive control (Fig. 5, colony 2). Based on colony color, the inhibition characteristics of eserine for displayed BmAChE were estimated for a concentration series of eserine solutions (1610 28 , 2610 28 , 3610 28 , 4610 28 , 5610 28 , 6610 28 and 7610 28 ). The B/B 0 decreased with increasing eserine concentrations (Fig. 6 (A)). The IC 50 of BmAChE was 4.17610 28 M. The results showed that the recombinant BmAChE that was displayed on the yeast surface exhibited high-sensitivity to eserine. Compared with the BmAChE expressed in Trichoplusia ni (BTI-Tn-5B1-4) cells [28], the sensitivity for eserine in our report was at about the same level, which indicated that BmAChE retained its natural activity after being displayed on the cell surface.

Detection of CB and OP Pesticides Using Displayed BmAChE
The displayed BmAChE enzyme was used to detect five CB and seven OP pesticides. Measurement of enzyme activity and inhibition studies were performed as described in the experimental section. The inhibition of BmAChE with CB pesticides is shown in Fig. 6 (B). An IC 50 of 1.92610 29 M was obtained with carbofuran, while an IC 50 of 1.13610 27 M was obtained with carbosulfan, 1.11610 28 M with isoprocarb, 6.58610 28 M with methiocarb and 6.41610 28 M with methomyl. Inhibition of BmAChE with seven OP pesticides is shown in Fig. 6 (C) and Table 2. Among them, trichlorphon showed the highest inhibitory effect on BmAChE activity with an IC 50 of 2.40610 27 M and a limit of detection of 3.89610 28 M. The maximum European Union (EU) residue limit was recently set at 0.01 mg/kg (approximately 4.0610 28 M) for pesticide residues in all agricultural products for   food or animal feed [29]. Therefore, the activity of the displayed BmAChE has sufficient sensitivity for the determination of most of the selected CB and OP pesticides. As seen in Table 2, for all five tested CB pesticides (carbofuran, carbosulfan, isoprocarb, methiocarb and methomyl), the sensitivity values of the displayed BmAChE were better than those of the common housefly (Musca domestica) and those of the common fruit fly (Drosophila melanogaster) AChEs. In addition, the sensitivity of our displayed BmAChE for the representative OP pesticides (dimethoate, isocarbophos and trichlorphon) is much better than the housefly AChE [30]. For dichlorphos, the sensitivity of the displayed BmAChE was at the same level as with the Drosophila melanogaster AChE [31], but a little less than that of the Bombyx mandarina AChE [32]. Further experimental optimization of the P. pastoris displayed BmAChE enzyme is expected to meet or exceed the pesticide detection requirements and the displayed BmAChE enzyme will be used for routine monitoring of CB and OP pesticides.
Analytical equipment-based methods typically used for the analysis of pesticides are not practicle enough to be used for simple, fast detection of large numbers of samples. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets [5]. Until now, most of the AChE enzymes used for the detection of pesticides have been extracted from fish and insect heads [33,34], requiring much preparation time, resulting in high costs for enzyme purification. However, the yeastdisplay technology has provided an alternative means for engineering a low-cost AChE enzyme with desirable activity and the developed cells can be immobilized by chemical methods or with physical methods for development of whole-cell biosensors [35]. Also, the yeast expression system is capable of folding and glycosylating heterologous eukaryotic proteins [36,37]. In particular, P. pastoris also has the advantage of high-density cultivation in inexpensive medium compared with other yeasts [38]. Therefore, the displayed AChE on the cell surface of P. pastoris potentially has many benefits and practical applications for pesticide detection.
AChE has been most often used for the detection of pesticides because of its broad-substrate specificity. In this study the AChE gene from Bombyx mori was cloned from a constructed vector and a P. pastoris cell surface display system was developed for the first time. The surface-displayed BmAChE was evaluated with eserine, and with CB and OP pesticides. The results demonstrated that the displayed BmAChE was bioactive and highly sensitive to CB and OP pesticides. The recombinant BmAChE surface-display can be used for detection of pesticide residues in AChE-based screening methods. Figure S1 Nucleic acid sequence of the 2900 bp fragment.

Supporting Information
(PDF)