Protocol

Ⅰ Bioinformatics Analysis

Before recombinant protein expression and analysis, it is necessary to understand the basic physicochemical properties of the protein. A number of online analysis websites can be used to analyze the relevant information of the protein.

Bioinformatics Analysis Tools

Ⅱ PCR

1 Basic PCR System

Using the pET46/LlADH plasmid as a template, and the primers designed above, the polymerase chain reaction was performed for site-directed mutagenesis of LlADH to construct a series of mutant plasmids.

2 Detection and Recovery of PCR Products

Prepare a 1% agarose gel to detect the success of PCR amplification. After confirming the successful PCR amplification, add DpnI enzyme and reaction buffer to eliminate the template DNA. Then, use the Cycle-Pure Kit product recovery kit from OMEGA to recover and purify the target fragment.

3 Transformation and Sequencing of PCR Products

1. In a ultra-clean workbench, take 10 μL of the recovered and purified target fragment and add it to 100 μL of competent cells of the cloning strain E. coli DH5α, and incubate on ice for 25 min
2. Place the competent cells containing the plasmid in a 42 ℃ water bath for heat shock for 1 min, then transfer to ice and let stand for 3 min
3. Add 700 μL of LB medium without antibiotics, place it in a shaker at 37 ℃ and 220 rpm, and culture for 40 min to resuscitate the bacterial cells
4. Centrifuge at 12000 rpm for 6 min, remove part of the supernatant, mix the remaining supernatant with the precipitate, and evenly spread it on the LB solid plate containing Amp resistance
5. Invert and place in a 37 ℃ incubator for 12-14 h of culture
6. After obvious single colonies grow on the plate, pick the single colonies and send them to Shanghai Shenggong Biological Engineering Co., Ltd. for sequencing

Ⅲ Protein Expression and Purification

1 Construction of Expression Strain

Transform the pET46/LlADH and a series of mutant plasmids with correct sequencing into the competent cells of the expression strain BL21(DE3), and the transformation steps are the same as those described above.

2 Protein Expression

The expression of LlADH and a series of mutant proteins was carried out using the same steps:

1. Pick the successfully transformed BL21(DE3) transformants, inoculate them into 6 mL of LB liquid medium containing Amp resistance, place them in a shaker, and culture at 220 rpm and 37 ℃ until the measured absorbance OD₆₀₀ reaches 0.6-0.8
2. Transfer the bacterial solution in Step 1 to 1 L of LB liquid medium containing Amp resistance, and continue the expanded culture at 37 ℃ and 220 rpm
3. When the absorbance OD₆₀₀ of the bacterial solution in Step 2 reaches 0.6-0.8, set the shaker temperature to 16 ℃, and take out 800 μL of the bacterial solution into an EP tube as a control group (without induction)
4. Except for the control group, add 0.4 mM IPTG to each 1 L of bacterial solution for induction, and continue the culture for 16-18 h
5. Use a floor centrifuge to centrifuge at 8000 rpm for 8 min to collect the bacterial cells, and resuspend them in Ni buffer A

3 Protein Purification

LlADH and a series of mutant proteins were all purified by the same method, i.e., one-step nickel column affinity chromatography. The specific steps are as follows:

1. After fully disrupting the resuspended bacterial cells using a low-temperature high-pressure cell disruptor (4 ℃, 1000 MPa), centrifuge the obtained bacterial solution with a floor centrifuge at 17000 rpm and 4 ℃ for 50 min to remove the precipitate
2. Load the nickel filler into the chromatography column, connect it to the AKTA protein purification system, set a flow rate of 4 mL/min, and rinse and compact the nickel filler in the chromatography column with ddH₂O
3. Rinse the nickel column with Ni buffer B at a flow rate of 4 mL/min for 15 min to remove the residual impurity proteins, and then rinse the nickel column with Ni buffer A at the same flow rate for 15 min to fill the nickel column and the AKTA protein purification system with a buffer suitable for the target protein
4. Load the supernatant centrifuged in Step 1 onto the nickel column via a pump at a flow rate of 3 mL/min. At this time, the target protein with a histidine tag can specifically bind to the nickel ions in the chromatography column filler. Then, rinse with Ni buffer A until the UV baseline is stable to remove the impurity proteins in the chromatography column that cannot bind to nickel ions
5. Utilize the property that the histidine tag in the target protein and imidazole compete to bind to nickel ions, use a 0-40% gradient elution within 40 min to effectively separate the target proteins with different binding capacities to the nickel filler in the chromatography column, and collect the eluent according to the UV curve detected by the AKTA protein system

4 Protein Purity Detection

Use SDS-PAGE gel electrophoresis to detect the purity of the target protein in the collected eluent:

1. Take 10 μL of the protein sample in the collection tube and mix it with 10 μL of 2×loading buffer
2. Insert the precast SDS-PAGE polyacrylamide gel into the electrophoresis tank, and pour an appropriate amount of SDS Running buffer
3. Use a pipette to add the samples into the sample wells respectively, and add 10 μL of tricolor pre-stained protein marker into one of the wells. Set the electrophoresis instrument parameters to 180 V for 36 min
4. After the electrophoresis is completed, disassemble the gel, mix the protein gel with the protein gel staining solution, heat for 40 s, and then incubate for 20 min
5. Pour off the staining solution, rinse with water to remove the residual staining solution, add the protein gel destaining solution, mix and heat for 40 s, incubate for 20 min, and repeat this step 2-3 times until the results are clearly visible

Ⅳ Protein Homogeneity Detection

1 Gel Filtration Chromatography

Gel filtration chromatography can separate substances with different molecular weights. The filler in the chromatography column is some inert porous network structure substances. Small-molecular-weight proteins can enter the interior of the porous network structure and require a longer elution time, while large-molecular-weight proteins are excluded from the porous network structure and require a shorter elution time. Thus, substances in the solution are separated according to their different molecular weights.

In this study, gel filtration chromatography was used to determine the molecular weight of the LlADH protein purified by nickel column affinity chromatography:

1. Collect the LlADH protein with high purity detected by SDS-PAGE, put it into a dialysis bag, and dialyze with Ni buffer A at 4 ℃ for 8 h to completely remove imidazole, and repeat this process 3 times
2. Put the dialyzed target protein into an ultrafiltration concentration tube with a molecular weight cutoff of 30 kDa for ultrafiltration concentration
3. Select a Superose™ 6 increase 10/300 GL column, connect it to the AKTA purification system, and rinse with Ni buffer A at a flow rate of 0.2 mL/min for 2 column volumes to equilibrate the chromatography column
4. Connect a 100 μL sample loop to the AKTA purification system, rinse it with ddH₂O and Ni buffer A in sequence, and then add a mixed protein sample with molecular weights of 669, 440, 158, 75, and 44 kDa
5. After setting the injection of the mixed protein sample in the sample loop, elute the chromatography column with Ni buffer A at a flow rate of 0.2 mL/min for 2 column volumes, observe and record the 5 peak positions and UV curves
6. Calculate the standard curve using the molecular weight and peak position of the mixed protein sample as parameters
7. Perform gel filtration chromatography on the LlADH protein in the same way, observe the UV curve, record the peak position of LlADH, and further calculate the molecular weight of LlADH

2 Negative Staining

Negative staining technology refers to mixing a heavy metal salt solution with a sample to make the sample show a good contrast. For protein samples stained by this method, the background is deeply stained, while the protein particles are lightly colored. Negative staining technology can be used to determine the state of the protein and its dispersion in the solution. Through the screening of negative staining electron microscopy, the particle composition and conformational homogeneity of the biological sample can be evaluated qualitatively.

Specific steps:

• Preparation of protein sample: Take one tube of protein from the single peak of gel filtration chromatography, centrifuge at 12000 rpm for 10 min at 4 ℃, and dilute the supernatant to 0.02 mg/mL with Ni buffer A for later use
• Hydrophilic treatment of negative staining grid: Set the parameters of the glow discharge instrument to 15 mA, GLOW for 25 s, and hold for 10 s. Place the grid in the glow discharge instrument, perform glow discharge according to the set parameters to make the grid charged, and the charged grid can better bind to the protein
• Preparation of negative staining sample:
  • Sampling: First, take 6 μL of protein sample and drop it on one side of the grid, let it stand for 1 min, and then absorb the residual sample with filter paper
  • Cleaning: Rinse the grid in Ni buffer A, absorb the liquid with filter paper, and repeat this twice
  • Primary staining: Add 6 μL of 2% uranyl acetate to the grid for primary staining of the sample, and after 10 s, absorb the residual sample with filter paper
  • Secondary staining: First, add 6 μL of 2% uranyl acetate for staining for 10 s, and absorb the residual sample with filter paper; finally, add 6 μL of 2% uranyl acetate for staining for 1 min, and absorb the residual sample with filter paper
  • Standing: Place the grid under a desk lamp until the liquid on its surface is completely dried, and then it can be used for observation
• Observation of negative staining sample: Use a 120 kV transmission electron microscope:
  • Send the grid into the lens barrel with a sample rod, ensure that the grid is correctly installed on the sample holder, and the lens is focused on the sample surface on the grid
  • First, set a low magnification of 100-200 times, observe the grid to ensure that it is not deformed or damaged, and then find the area on the grid where the carbon film is complete and the staining is uniform
  • Increase the magnification to 2600 times, adjust the Z-high to align the electron beam with the sample surface
  • To facilitate the capture of clear images, increase the contrast to evaluate the dispersion state of the protein in the solution, increase the magnification to 92000 times, and set the defocus amount between -1 and -2 μm

3 Static Light Scattering

In this experiment, the dynamic light scattering (DLS) method was used, and NanoBrook 90 Plus was used to detect the particle size and distribution of LlADH multimers.

Experimental steps:

1. Preparation of protein sample: Filter one tube of protein from the single peak of gel filtration chromatography with a 0.22 μm filter membrane, and dilute it with Ni buffer A
2. Instrument parameter setting: Set the incident light wavelength of the laser emitter to 659 nm, the incident angle to 90°, the measurement temperature to 4 ℃, and the measurement time to 3 min. Perform the measurement 3 times in total, and take the average value of the results

Ⅴ Cryo-Sample Preparation

Since the breakthrough of cryo-electron microscopy technology in recent years (direct electron detector, image drift correction, three-dimensional reconstruction technology, etc.), cryo-electron microscopy technology has become an important structural analysis technology in the field of structural biology. The first step is to prepare the cryo-sample.

Specific process:

1. Preparation of protein sample: Centrifuge the protein from the single peak of gel filtration chromatography at 12000 rpm for 10 min at 4 ℃ to remove the precipitate, dilute it to 0.5 mg/mL with Ni buffer A, and place it on ice
2. Hydrophilic treatment of cryo-grid: Perform glow discharge hydrophilic treatment on the copper grid (R1.2/1.3, 300 mesh) for cryo-electron microscopy, and set the parameters to 15 mA, GLOW for 25 s, and hold for 10 s
3. Pre-cooling of grid box: Put the grid box into liquid nitrogen, and a solid-liquid mixture of liquid ethane should be formed in the central metal groove of the grid for sample preparation
4. Preparation of cryo-sample: Drop 5 μL of protein onto the grid treated with glow discharge, set the temperature at 4 ℃ and humidity at 100%, set the cryo-sample preparation conditions (blot-time: 5 s, wait-time: 5 s, blot-force: 0 s), and after completion, put the grid into the solid-liquid mixture of liquid ethane for cooling
5. Storage of sample: Place the grid box in a centrifuge tube stored in a liquid nitrogen tank, and do not leave the liquid nitrogen throughout the process

Ⅵ Cryo-Sample Data Collection and Processing

1 Cryo-Sample Data Collection

The cryo-electron microscopy data were collected at the Cryo-Electron Microscopy Facility Platform of Hubei University. A 300 kV Titan Krios transmission electron microscope equipped with a Gatan K3 Summit direct electron detector was used. Data collection was performed at a magnification of 105,000, corresponding to a final pixel size of 0.425 Å on the image. Under the conditions of a dose rate of 15.66 e⁻/pixel/s and a total exposure time of 2.5 s, the photos were divided into 40 frames, and the total electron dose of each frame of image was 52 e⁻/Ų. The defocus value range was -1.0 ~ -2.4 μm.

2 Cryo-Sample Data Processing

Use cryoSPARC v3.3.1 software to perform offset correction and contrast transfer function evaluation on the original images, then automatically select protein particles for two-dimensional classification. Select some good particles to construct an initial template, and use this as a template for a new round of two-dimensional classification. The selected protein particles are used for further three-dimensional classification. After multiple rounds of optimization, the final protein electron density map is reconstructed.

Ⅶ Structure Refinement

The structure of LlADH was built using the structure with PDB ID 8IHQ (with 76% structural similarity to LlADH) in the PDB database as the original model. The UCSF Chimera software was used to superimpose the 8IHQ structure with the electron density map obtained from the electron microscopy data processing. The atomic model of the LlADH protein was built in COOT, and various building tools in COOT were used to correct the amino acids with non-standard parameters, so that all parameters of the built structure reached the standard level. Finally, the Real-spacerefinement function in the Phenix software was used for spatial refinement, and the MolProbity function was used for the final evaluation of the model. The structure and mapping were completed using PyMol and UCSF Chimera software.

Ⅷ Enzyme Activity Analysis

Enzyme activity refers to the catalytic ability of an enzyme in a certain chemical reaction, and the level of enzyme activity can be expressed by the conversion rate of the enzyme-catalyzed chemical reaction under certain conditions. In this experiment, the enzyme activity level was compared by measuring the consumption of substrates, the conversion of reaction intermediates, and the production of products.

1 Enzyme Concentration Quantification

To reduce experimental errors, the BCA method was used to quantify the enzyme concentration. In this method, different concentrations of BCA standard and their corresponding absorbance values at 595 nm were used as the horizontal and vertical coordinates, respectively, to draw a standard curve. Then, the samples were measured in the same way, the enzyme concentration was calculated from the standard curve, and the enzyme was diluted and adjusted to 1 mg/mL with Ni buffer A for subsequent reactions.

2 Enzyme Reaction System

Specific experimental procedures:

• Comparison of enzyme activities between LlADH and ADH3: The reaction system was as shown in Table above. 5 μg of LlADH and ADH3 were mixed with 10 μg of OTA in 20 mM Tris-HCl buffer (pH 8.0), and reacted in a metal bath at 40 ℃ for 20 min. After the reaction, an equal volume of acetonitrile was added to terminate the reaction. The results were compared with the enzyme activity of ADH3 set as 100%.
• Effect of temperature on enzyme activity: The reaction system was as shown in Table above. 5 μg of LlADH was mixed with 10 μg of OTA in 20 mM Tris-HCl buffer (pH 8.0), and reacted in a metal bath at temperatures ranging from 0 to 80 ℃ for 20 min. The results were compared with the enzyme activity at the optimal temperature set as 100%.
• Effect of pH on enzyme activity: The reaction system was as shown in Table above. 5 μg of LlADH was mixed with 10 μg of OTA in 20 mM citrate-phosphate buffer (pH 3.0-7.0), 20 mM Tris-HCl buffer (pH 7.0-9.0), and 20 mM glycine-NaOH buffer (pH 9.0-11.0), respectively, and reacted in a metal bath at 40 ℃ for 20 min. The results were compared with the enzyme activity at the optimal pH set as 100%.
• Effect of different metal ions on enzyme activity: The reaction system was as shown in Table above. 5 μg of LlADH was mixed with 10 μg of OTA in 20 mM Tris-HCl buffer (pH 8.0), and 10 mM LiCl, CaCl₂, MgCl₂, CoCl₂, MnCl₂, NiCl₂, FeCl₂, FeCl₃, CuCl₂, and ZnCl₂ were added to the system. The reaction was carried out in a metal bath at 40 ℃ for 20 min. The results were compared with the enzyme activity of the protein without added metal ions set as 100%.
• Thermal stability test: Place the enzyme in a pre-reaction at temperatures ranging from 0 to 80 ℃ for 1 h. The reaction system was as shown in Table above. 5 μg of LlADH was mixed with 10 μg of OTA in 20 mM Tris-HCl buffer (pH 8.0), and reacted in a metal bath at 40 ℃ for 20 min. Determine the residual enzyme activity of LlADH, and the results were compared with the enzyme activity of the protein with the best thermal stability set as 100%.
• Enzyme activity test of different mutants: The reaction system was as shown in Table above. 5 μg of LlADH and mutants were mixed with 10 μg of OTA in 20 mM Tris-HCl buffer (pH 8.0), and reacted in a metal bath at 40 ℃ for 20 min. The results were compared with the enzyme activity of the wild-type LlADH set as 100%.

3 Enzyme Reaction Analysis

In this experiment, high-performance liquid chromatography (HPLC) was used to analyze and calculate the material changes before and after the enzyme reaction:

1. All enzyme reactions were repeated 3 times, and the results were expressed as mean ± standard deviation. After the reaction, an equal volume of chromatographic grade acetonitrile was immediately added to quench the reaction. The reacted liquid was filtered through a 0.22 μm filter membrane and prepared for injection analysis
2. Prepare the mobile phase buffer HPLC buffer. Pumps A and B were filled with ddH₂O and 95% acetonitrile + 5% glacial acetic acid, respectively, and used after ultrasonic degassing
3. Install the InerSustain C18 column (4.6×250 mm, 5 μm) on the HPLC. Use Shimadzu CMB-20A, and the system detector is SPD-M20A photodiode array detector
4. Take 20 μL of sample for injection analysis, perform linear gradient elution with 50% HPLC buffer B within the range of 0-25 min, with a detection wavelength of 330 nm, a flow rate of 1.0 mL/min, and a column oven temperature of 40 ℃
5. At the maximum absorption wavelength, calculate the enzyme reaction results by integrating the peak area

References

1. Chebil S, Rjiba-Bahri W, Oueslati S, et al. Ochratoxigenic fungi and ochratoxin A determination in dried grapes marketed in Tunisia[J]. Annals of Microbiology, 2020, 70: 1-9.
2. Heintz M M, Doepker C L, Wikoff D S, et al. Assessing the food safety risk of ochratoxin A in coffee: A toxicology‐based approach to food safety planning[J]. Journal of Food Science, 2021, 86(11): 4799-4810.
3. Delgado-Ospina J, Molina-Hernandez J B, Viteritti E, et al. Advances in understanding the enzymatic potential and production of ochratoxin A of filamentous fungi isolated from cocoa fermented beans[J]. Food Microbiology, 2022, 104: 103990