Insect atp-binding cassette superfamily transporter protein and use thereof

Abstract

The present application relates to an insect ATP-binding cassette superfamily transporter protein and its application. The insect ATP-binding cassette superfamily transporter protein is a homodimer, each monomer having an ATP-binding and hydrolyzing domain, a transmembrane domain containing six transmembrane helices and an extracellular domain; in the transmembrane domain of the protein structure, a "arched" substrate binding channel is formed by helix 1, helix 2 and helix 5 of each monomer. The experiments of the present application show that the ATP-binding cassette superfamily transporter protein binds with a small pesticide molecule substrate in the "arched" substrate binding channel and discharges outside the body, thereby producing drug resistance, so that the insect ATP-binding cassette superfamily transporter protein can be used as a target for insecticide resistance to design efficient inhibitor compounds and can be used as a target to solve insecticide resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an insect adenosine triphosphate binding cassette superfamily transporter and its applications. Background Technology

[0002] Agricultural pests and diseases are a significant limiting factor for sustainable agricultural development. my country has over 730 common agricultural pests, more than 20 of which are major pests, causing crop yield losses of over 30% during severe outbreaks. Currently, chemical pesticide control remains the most important and widespread strategy for controlling most pests. However, recent studies have shown that many insects have evolved multiple resistance mechanisms, developing resistance to a range of insecticides. Research on agricultural pest resistance mechanisms is of significant theoretical importance for the rational use of pesticides and effective pest control. Adenosine triphosphate (ATP) binding cassette (ABC) transporters are one of the largest transmembrane protein families in organisms, utilizing ATP hydrolysis to provide energy for the transmembrane transport of various substances. Based on sequence similarity and conserved domains, the ABC transporter family can be divided into eight subfamilies (ABCA-ABCH), each with different numbers of members and functions. These proteins are distributed in various organisms, and their main functions include transporting substances, signal transduction, acting as cell surface receptors, and participating in intracellular DNA repair, transcription, and regulating gene expression. Recent studies have shown that mutations or overexpression of ABC transporters are not only closely related to arthropod resistance to chemical pesticides, but also play a crucial role in resistance to Bt toxins. The ABCH subfamily of transporters are arthropod-specific ABC transporters and can serve as potential targets for resistance screening. Elucidating the mechanism by which pests transport pesticide molecules extracellularly via ABCH transporters is of great significance for addressing pesticide resistance issues. Summary of the Invention

[0003] In view of the needs of existing technology, this invention provides a method for screening ABCH transporter protein pesticide molecular substrates based on ATPase activity. The cryo-electron microscopy structure of the ABCH superfamily transporter protein and its complex with pesticide small molecules, obtained through three-dimensional structural analysis and structural information, can be used for insecticide design and has broad application prospects in the field of pest control.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0005] The insect adenosine triphosphate binding cassette superfamily transporter ABCH has a three-dimensional structure, in which the protein structure is a homodimer with C2 symmetry. Each monomer of the protein structure has an adenosine triphosphate binding and hydrolysis domain, a transmembrane domain containing 6 transmembrane helices, and an extracellular domain. In the transmembrane domain of the protein structure, helices 1, 2, and 5 of each monomer form an "arched" substrate binding channel.

[0006] The helical sequences of the “arched” substrate binding channel are as follows: Helix 1 sequence is NVGVMLFIFALPVMQVILFCLAI, Helix 2 sequence is FTDFVAPGVILTIVFFLAVALTSSALIIER, and Helix 5 sequence is ERNAIQLALGSFYPTLLL.

[0007] The insect adenosine triphosphate binding cassette superfamily transporter ABCH is described in the insect Tribolium castaneum Herbst. The amino acid sequence of the insect adenosine triphosphate binding cassette superfamily transporter is shown in SEQ ID No. 1.

[0008] The preparation method of insect adenosine triphosphate binding cassette superfamily transporter ABCH includes the following steps:

[0009] 1) Expression and purification of ABCH protein

[0010] ABCH protein was recombinantly expressed in HEK293F cells. After the collected cells expressing ABCH were lysed, high-purity ABCH protein was obtained by affinity chromatography and gel filtration chromatography.

[0011] 2) Cryo-electron microscopy sample preparation

[0012] (1) Concentrate high-purity ABCH protein to 5 mg / mL; (2) Perform glow discharge treatment for 30 seconds on a Quantifoil R1.2 / 1.3 300-mesh gold grid, using H2 and O2 as the glow discharge gases; (3) Prepare the sample using FEI's Vitrobot Mark IV, setting the freezing conditions as follows: blot-time: 3s, wait-time: 5s, drain-time: 0s, blot-force: 3; (4) Take 3 μl of protein and add it to the grid surface. After the Vitrobot Mark IV finishes the sample preparation process, quickly place the sample into liquid ethane and then store it in liquid nitrogen.

[0013] 3) Data was collected and analyzed from frozen samples under a cryo-electron microscope to obtain the three-dimensional structure of protein ABCH.

[0014] In step 1), the recombinant expression of ABCH protein was carried out using the expression vector pcDNA3.1. The recombinant plasmid was transfected into HEK293F cells using the linear PEI transfection method, and the protein was expressed under the conditions of 37℃, 150rpm, and 5% CO2.

[0015] The method for preparing high-purity ABCH protein in step 1) includes the following steps:

[0016] (A) HEK293F cells containing ABCH protein particles were collected and suspended in a buffer solution of pH 8.0, 20 Mm Tris-HCl, and 150 Mm NaCl. The cells were then homogenized using a high-pressure homogenizer and centrifuged at 13,000 rpm for 30 min. The supernatant was collected and ultracentrifuged at 150,000 g to collect the cell membrane. The cell membrane was then dissolved in a buffer solution of pH 8.0, 20 Mm Tris-HCl, 150 Mm NaCl, and 1% Glyco-diosgenin for 1 h to fully dissolve the target protein on the cell membrane. The mixture was then ultracentrifuged at 150,000 g to collect the supernatant.

[0017] (B) The target protein was enriched by Strep affinity chromatography on the supernatant fraction. After the supernatant flowed through the Strep affinity chromatography column, it was washed with buffer containing pH 8.0, 20Mm Tris-HCl, 150Mm NaCl, and 1% Glyco-diosgenin to remove impurities. Then, the target protein was immediately eluted with buffer containing pH 8.0, 20Mm Tris-HCl, 150Mm NaCl, 1% Glyco-diosgenin, and 50mM biotin.

[0018] (C) After collecting the protein, concentrate it to 500 μl and further purify it using a Superose 6 gel filtration chromatography column to obtain the target protein in high purity.

[0019] Application of insect adenosine triphosphate binding cassette superfamily transporter ABCH in screening small molecule pesticide substrates.

[0020] Application of insect adenosine triphosphate binding cassette superfamily transporter ABCH as an insecticide resistance target.

[0021] The application according to claim 8 includes the following steps:

[0022] (a) Based on the principle of substrate activation of ATP hydrolysis activity, small molecule pesticide substrates will be screened for insect adenosine triphosphate binding cassette superfamily transporter ABCH.

[0023] (b) Preparation of composite cryo-electron microscopy samples

[0024] (I) Concentrate high-purity ABCH protein to 5 mg / mL and add a small molecule pesticide substrate to a final concentration of 100 μM; (II) Perform glow discharge treatment for 30 seconds using a Quantifoil R1.2 / 1.3 300-mesh gold grid with H2 and O2 as the glow discharge gases; (III) Prepare the sample using the FEI Vitrobot Mark IV with the following freezing conditions: blot-time: 3 s, wait-time: 5 s, drain-time: 0 s, blot-force: 3; (IV) Take 3 μl of protein and add it to the grid surface. After the Vitrobot Mark IV finishes the sample preparation process, quickly place the sample into liquid ethane and then store it in liquid nitrogen.

[0025] (c) Data were collected and analyzed from frozen samples under a cryo-electron microscope to obtain the three-dimensional structure of protein ABCH and the binding site of small molecule pesticide substrate;

[0026] (d) Using the "arched" channels in the three-dimensional structure and the small molecule pesticide substrate binding sites as targets to guide the design and development of drug resistance reversal agents targeting ABCH.

[0027] The small molecule pesticide substrate is phenoxycarb, and the binding sites are Arg404, Met405, Val411, Phe414, Phe608, Ser616, Ile707, Gln708, and Ala710.

[0028] Based on the principle of substrate activation of ATP hydrolysis activity, this invention screened phenoxycarb as a potential pesticide substrate for ABCH by detecting the activation effect of 15 pesticide small molecules on the ATPase activity of ABCH.

[0029] The three-dimensional structures of the insect adenosine triphosphate binding cassette superfamily transporter ABCH and the ABCH-phenoxycarb complex were resolved using cryo-electron microscopy. The structures are three-dimensional, homodimers with C2 symmetry. Each monomer of the protein structure has an adenosine triphosphate binding and hydrolysis domain, a transmembrane domain containing six transmembrane helices, and an extracellular domain. Within the transmembrane domain of the protein structure, helices 1, 2, and 5 of each monomer form an "arched" substrate-binding channel. The helical sequence of this "arched" substrate-binding channel is: NVGVMLFIFALPVMQVILFCLAI (helix 1), FTDFVAPGVILTIVFFLAVALTSSALIIER (helix 2), and ERNAIQLALGSFYPTLLL (helix 5). The two intracellular ends of this "arched" substrate binding channel each bind one molecule of phenoxycarb. This binding pocket is composed of Arg404, Met405, Val411, Phe414, Phe608, Ser616, Ile707, Gln708, and Ala710. Figure 5 ).

[0030] This invention also evaluated the efflux activity of ABCH against phenoxycarb. Cells transfected with ABCH or EGFP (control group) were cultured for 24 hours, then phenoxycarb was added, and after another 72 hours of culture, cell viability was assessed using a CCK8 assay. The results showed that cells transfected with ABCH exhibited a significant detoxification ability against phenoxycarb, indicating that ABCH can efflux phenoxycarb (…). Figure 7 ).

[0031] This invention focuses on the ABCH superfamily transporter protein of erythropoietin triphosphate binding cassette and provides an efficient and universal method for the expression and preparation of recombinant proteins based on a two-step method of affinity chromatography and gel sieving. High-purity proteins are obtained, and cryo-electron microscopy samples are prepared. The complete structure of this protein is resolved by cryo-electron microscopy.

[0032] This application, based on the screening mechanism of substrate activation of ATP hydrolysis activity, discovered that phenoxycarb molecule may be a substrate of ABCH. Further analysis of the structure of the ABCH-phenoxycarb complex revealed the presence of phenoxycarb in the substrate transport channel, indicating that phenoxycarb is indeed a substrate of ABCH. Simultaneously, cytotoxic efflux experiments demonstrated that ABCH possesses detoxification capabilities against phenoxycarb, meaning that ABCH mediates insecticide resistance formation by effluxing phenoxycarb. The insect adenosine triphosphate binding cassette superfamily transporter ABCH has a narrow, arched substrate-binding channel. Pesticide substrates bind to the transporter ABCH through this channel and are then expelled from the body. Therefore, this channel can serve as a target for insecticide resistance, guiding the design and development of highly effective inhibitors.

[0033] Furthermore, the application method provided by this invention can be extended to all members of the ABC transporter family. Attached Figure Description

[0034] Figure 1 The results show the purification of the ABCH recombinant protein. The curve is the elution curve of gel filtration chromatography, and the electrophoresis sample in SDS-PAGE is the protein sample after gel filtration purification.

[0035] Figure 2 The overall structure of the ABCH and phenoxycarb complex is shown as a ribbon model, with the phenoxycarb molecule shown as a ball-and-stick model, and boxes indicating the approximate location of the cell membrane.

[0036] Figure 3 The "arched" channel is a structural feature derived from the spirals 1, 2, and 5 of two individual units, which form an "arched" channel that appears as a mesh pattern.

[0037] Figure 4 The protein profile shows the location of the "arched" channel in the phenoxycarb binding mode. At the intracellular end, two phenoxycarb molecules are bound, and the phenoxycarb molecules are shown as a ball-and-stick model.

[0038] Figure 5 Amino acid residues that form the phenoxycarb binding pocket.

[0039] Figure 6 The results show the screening results for ABCH insecticide substrates.

[0040] Figure 7 The individual electron microscopy structures of ABCH are displayed as a ribbon model.

[0041] Figure 8 The ordinate represents the detoxification activity of ABCH against phenoxycarb, and the vertical axis represents the percentage of cell viability. 1 - untransfected cells; 2 - untransfected cells treated with phenoxycarb; 3 - cells transfected with EGFP; 4 - cells transfected with EGFP treated with phenoxycarb; 5 - cells transfected with ABCH; 6 - cells transfected with ABCH treated with phenoxycarb. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0043] All reagents used in the examples are commercially available.

[0044] The amino acid sequence of the adenosine triphosphate binding cassette superfamily transporter ABCH selected by the present invention is shown in SEQ ID No. 1.

[0045] Example 1: Preparation method of insect adenosine triphosphate binding cassette superfamily transporter ABCH

[0046] 1. Construction of ABCH expression vector

[0047] (1) A HindIII restriction site sequence and a Twin-strep purification tag were inserted at the N-terminus of the ABCH protein, and an XhoI restriction site sequence was inserted at the C-terminus. Codon optimization was performed for HEK293 cells to synthesize the full-length protein gene sequence, as shown in SEQ ID No. 2.

[0048] (2) The full-length gene sequence and pcDNA3.1 were digested with HindIII and XhoI restriction enzymes and then ligated with T4 DNA ligase to obtain the recombinant expression vector. The expression vector was successfully constructed by double enzyme digestion and plasmid sequencing.

[0049] (3) The recombinant plasmid was transformed into Escherichia coli DH5α competent cells by chemical transformation to obtain cloned strains.

[0050] 2. Expression of ABCH protein

[0051] (1) Plasmid extraction

[0052] The cloned strain was cultured overnight at 37°C in LB medium. High-concentration, high-purity recombinant plasmids were extracted using a plasmid extraction kit (Tiangen Biotech) and stored at -20°C for later use.

[0053] (2) Resuscitation and passage of HEK293F cells

[0054] HEK293F cells were removed from liquid nitrogen and quickly thawed in a 37°C water bath for approximately 2 minutes. After thawing, the cells were immediately transferred to preheated culture medium and cultured at 150 rpm, 37°C, and 5% CO2. When the cell density reached 3 × 10⁻⁶ cells / year... 6 Then, the cells were passaged, with the initial passage density controlled at 0.2-0.3 × 10⁻⁶. 6 .

[0055] (3) HEK293F cell transfection

[0056] HEK293F cells were transfected using a linear PEI cationic polymer transfection method. The transfection method is as follows:

[0057] Before transfection, the cell density was diluted to 1×10⁻⁶ cells with culture medium. 6 Continue culturing for 3-4 hours or overnight, maintaining a density of 2×10⁻⁶. 6 Within 2 μg / ml of culture medium, add the plasmid to 10 ml of preheated fresh culture medium, vortex to mix. Take 3 times the volume of PEI solution and add it to the culture medium containing the plasmid, adding it slowly dropwise while vortexing. After adding all the plasmid, vortex vigorously for 10 seconds and let it stand for 30 minutes to allow the PEI-DNA complex to form. After standing, slowly add the PEI-DNA complex culture medium solution to the cultured cells while shaking the flask to mix thoroughly. Then, culture at 150 rpm, 37°C, and 5% CO2 for 2 days. Finally, centrifuge at 1000g for 5 minutes to collect the cells.

[0058] 3. Purification of ABCH protein

[0059] (1) Collected HEK293F cells were resuspended in 20 Mm Tris-HCl (pH 8.0), 150 Mm NaCl buffer, and homogenized using a high-pressure homogenizer. The cells were then centrifuged at 13000 rpm for 30 min, and the supernatant was collected. The cell membranes were collected by ultracentrifugation at 150000 g. The cell membranes were then homogenized in 20 Mm Tris-HCl (pH 8.0), 150 Mm NaCl, 1%...

[0060] Dissolve the target protein on the cell membrane for 1 hour under Glyco-diosgenin conditions to ensure complete dissolution. Then, centrifuge the mixture at 150,000g and collect the supernatant.

[0061] (2) The target protein was enriched by Strep affinity chromatography on the supernatant fraction. After the supernatant flowed through the Strep affinity chromatography column, it was washed with buffer containing 20 Mm Tris-HCl (pH 8.0), 150 Mm NaCl, and 1% Glyco-diosgenin. Then, the target protein was immediately eluted with buffer containing 20 Mm Tris-HCl (pH 8.0), 150 Mm NaCl, 1% Glyco-diosgenin, and 50 mM biotin.

[0062] (3) After collecting the protein, it was concentrated to 500 μl and further purified using a Superose 6 gel filtration chromatography column to obtain high-purity target protein. Through the above purification steps, high-purity ABCH protein was obtained (see attached results). Figure 1 It can be used for the preparation of cryo-electron microscopy samples.

[0063] Example 2: Screening of pesticide small molecule substrates for insect adenosine triphosphate binding cassette superfamily transporter ABCH

[0064] The ATP hydrolytic activity of ABCH was determined using a pyruvate-coupled experimental method. This experiment is based on a reaction system capable of regenerating hydrolyzed ATP while producing pyruvate. After each ATP hydrolysis cycle catalyzed by ABCH, a regeneration system containing phosphoenolpyruvate (PEP) and pyruvate kinase (PK) converts one molecule of PEP into pyruvate, thereby allowing ADP to be converted back into ATP. Pyruvate reacts with 2,4-dinitrophenylhydrazine to form acetone-2,4-dinitrophenylhydrazone, which can be quantified by measuring the absorbance at 450 nm, indicating that the amount of pyruvate produced equals the amount of ATP consumed.

[0065] The ATPase activity of purified ABCH in detergent was determined at 30°C. First, ABCH was incubated with different small pesticide molecules for 10 minutes each, with a control sample containing no small pesticide molecule. The incubation system contained 50 mM Tris (pH 8.0), 2 mM MgCl2, 150 mM KCl, 1 mM DTT, and 0.03% Glyco-diosgenin. Then, 5 mM PEP, 20 U / ml pyruvate kinase, and 2 mM ATP were added. After 30 minutes, the reaction was stopped by adding 100 μL of a solution containing 0.36 mM 2,4-dinitrophenylhydrazine and 0.4 M HCl, and the mixture was incubated at 37°C for 15 minutes. Finally, 100 μL of 2.5 M NaOH and 0.1 M EDTA were added to each reaction, and the absorbance at 450 nm was recorded using a 96-well plate. ATPase activity was calculated using a pyruvate standard curve. By comparing the activation effects of different pesticide small molecules on ABCH ATPase activity, potential pesticide small molecule substrates for ABCH were identified (screening results are shown in [link to study]). Figure 6 ).

[0066] The results of ATPase-based insecticide substrate screening showed that phenoxycarb significantly stimulated the ATPase activity of ABCH, indicating that phenoxycarb is a potential insecticide substrate for ABCH.

[0067] Example 3: Preparation of frozen samples of *Triplophysa rubrum* ABCH and its complex with phenoxycarb.

[0068] (1) In 50mM Tris (pH 8.0), 150mM NaCl, 2mM DTT, 2mM Mg 2+ High-purity ABCH protein was obtained under 0.05% Glyco-diosgenin conditions and concentrated to 5 mg / mL;

[0069] (2) Quantifoil's R1.2 / 1.3, 300-mesh gold grid was subjected to glow discharge for 30 seconds. The glow discharge gas was H2 and O2.

[0070] (3) Sample preparation was performed using the Vitrobot Mark IV from FEI, and the freezing conditions were set (blot-time: 3s, wait-time: 5s, drain-time: 0s, blot-force: 3).

[0071] (4) Take 3 μl of protein and add it to the surface of the grid. After the Vitrobot Mark IV finishes the sample preparation procedure, quickly place the sample into liquid ethane and then store it in liquid nitrogen.

[0072] Frozen samples of the ABCH and phenoxycarb complex were prepared using the same method, except that in step (1), after protein concentration, phenoxycarb (dissolved in DMSO) was added to a final concentration of 100 μM, and after incubation for 30 minutes, the sample was prepared using the same method.

[0073] Example 4: Structural analysis of *Triplophysa rubrum* ABCH alone and its complex with phenoxycarb.

[0074] Frozen samples were first screened using a 200kV cryo-electron microscope, followed by data collection under a 300kV cryo-electron microscope. The electron microscopes initially used for data collection were the FEI Titan Krios and the Gatan K2 Summit direct electron detection camera. Data collection was performed using Serial EM software in low-dose mode with automatic imaging. The magnification was set to 22500x, corresponding to a pixel size of [missing information]. Total electron dose is The underfocus value range is set to -1 to -2 μm.

[0075] For the ABCH sample alone, a total of 5954 single-particle images were collected. The image data were processed using cryoSPARC software to obtain a resolution of [resolution missing]. The electron density at resolution was determined. For the ABCH and phenoxycarb complex sample, a total of 5243 single-particle images were collected. The image data were processed using cryoSPARC software to obtain an electron density at a resolution of [resolution value missing]. The electron density at resolution was determined, and then the structure was constructed and finely optimized using PHENIX and Coot based on the protein sequence. The structure was evaluated by PDB Validation Service (see Table 1 for structural parameters).

[0076] Table 1. Statistics of cryo-electron microscopy data collection and structure correction data

[0077]

[0078] The obtained ABCH structure is a first H subfamily adenosine triphosphate binding cassette superfamily transporter. This protein structure is a homodimer, with each monomer containing an intracellular nucleotide-binding domain (NBD), an extracellular domain (ECD), and a transmembrane domain containing six transmembrane helices (TMD). Figure 2 , Figure 7 The TMD domain of this dimer protein forms an "arched" channel, which is formed by helix 1, helix 2, and helix 5 of the TMD domain. Figure 3 The helical sequences involved are NVGVMLFIFALPVMQVILFCLAI (helix 1), FTDFVAPGVILTIVFFLAVALTSSALIIER (helix 2), and ERNAIQLALGSFYPTLLL (helix 5). Two phenoxycarb binding pockets are formed at the two ends of the "arched" channel near the intracellular side. Figure 4 The combined pocket is composed of Arg404, Met405, Val411, Phe414, Phe608, Ser616, Ile707, Gln708, and Ala710. Figure 5 The acquisition of these structural details can be applied to the design of structure-based drug resistance reversal agents.

[0079] Example 5: Detoxification experiment of ABCH on phenoxycarb

[0080] This experiment used the HEK293F cell line to evaluate its ability to detoxify ABCH. First, HEK293 cells were cultured in medium to a density of 1.5 × 10⁻⁶. 6 The density was determined, and then HEK293F cells were transfected with ABCH and EGFP control plasmids using linear PEI 40000 transfection reagent. After culturing for 24 hours to allow the transfected proteins to express, 50 nM phenoxycarb was added and the cells were cultured for another 72 hours. Cell viability was then detected using the CCK8 assay kit.

[0081] The results showed that cells transfected with ABCH exhibited a significant detoxification ability against phenoxycarb, indicating that ABCH can detoxify phenoxycarb (…). Figure 8 )

[0082] The detoxification effect of ABCH on phenoxycarb, combined with the structural information of the ABCH-phenoxycarb complex, can demonstrate that ABCH recruits phenoxycarb molecules into the "arched" substrate transport channel, thereby achieving the efflux of phenoxycarb and mediating the generation of phenoxycarb resistance. Therefore, the "arched" channel and the phenoxycarb binding site can serve as targets to guide the design and development of drug resistance reversal agents targeting ABCH.

Claims

1. Application of insect adenosine triphosphate binding cassette superfamily transporter ABCH in screening small molecule pesticide substrates, wherein the small molecule pesticide substrate is phenoxycarb; the amino acid sequence of the insect adenosine triphosphate binding cassette superfamily transporter is shown in SEQ ID No.

1.

2. The application according to claim 1, comprising the following steps: (a) Based on the principle of substrate activation of ATP hydrolysis activity, small molecule pesticide substrates were screened from the insect adenosine triphosphate binding cassette superfamily transporter ABCH. (b) Preparation of composite cryo-electron microscopy samples (I) Concentrate high-purity ABCH protein to 5 mg / mL and add a small molecule pesticide substrate to a final concentration of 100 μM; (II) Perform glow discharge treatment for 30 seconds using a Quantifoil R1.2 / 1.3 300-mesh gold grid with H2 and O2 as the glow discharge gases; (III) Prepare the sample using the FEI Vitrobot Mark IV with the following freezing conditions: blot-time: 3 s, wait-time: 5 s, drain-time: 0 s, blot-force: 3; (IV) Take 3 μl of protein and add it to the grid surface. After the Vitrobot Mark IV finishes the sample preparation process, quickly place the sample into liquid ethane and then store it in liquid nitrogen. (c) Data were collected and analyzed from frozen samples under a cryo-electron microscope to obtain the three-dimensional structure of protein ABCH and the binding site of small molecule pesticide substrate; The small molecule pesticide substrate is phenoxycarb, and the binding sites are Arg404, Met405, Val411, Phe414, Phe608, Ser616, Ile707, Gln708, and Ala710.

3. The application according to claim 2, wherein the method for preparing the high-purity ABCH protein is to recombinantly express ABCH protein using HEK293F cells, and after lysing the collected ABCH-expressing cells, obtain high-purity ABCH protein using affinity chromatography and gel filtration chromatography.

4. The application according to claim 3, wherein the recombinant expression of ABCH protein is performed by transfecting the recombinant plasmid into HEK293F cells using the expression vector pcDNA3.1 and the protein expression is carried out at 37°C, 150 rpm, and 5% CO2.

5. The application according to claim 4, wherein the method for obtaining high-purity ABCH protein comprises the following steps: (A) The collected HEK293F cells containing ABCH protein particles were suspended in a buffer solution of pH 8.0, 20Mm Tris-HCl, and 150Mm NaCl. The cells were then homogenized using a high-pressure homogenizer and centrifuged at 13,000 rpm for 30 min. The supernatant was collected and ultracentrifuged at 150,000 g to collect the cell membrane. The cell membrane was then dissolved in a buffer solution of pH 8.0, 20Mm Tris-HCl, 150Mm NaCl, and 1% Glyco-diosgenin for 1 h to fully dissolve the target protein on the cell membrane. The mixture was then ultracentrifuged at 150,000 g to collect the supernatant. (B) The target protein was enriched by Strep affinity chromatography on the supernatant fraction. After the supernatant flowed through the Strep affinity chromatography column, it was washed with buffer containing pH 8.0, 20Mm Tris-HCl, 150Mm NaCl, and 1% Glyco-diosgenin to remove impurities. Then, the target protein was immediately eluted with buffer containing pH 8.0, 20Mm Tris-HCl, 150Mm NaCl, 1% Glyco-diosgenin, and 50mM biotin. (C) After collecting the protein, concentrate it to 500 μl and further purify it using a Superose 6 gel filtration chromatography column to obtain the target protein with high purity.

Images