A purification method for recombinant canine CYP450 protein

Abstract

This invention relates to a method for purifying recombinant canine CYP450 protein. The method includes the following steps: S01. Separating and extracting total membrane proteins from the fermentation broth of recombinant yeast expressing recombinant canine CYP450 protein; S02. Subjecting the total membrane proteins to gradient washing, denaturation dissolution, dialysis refolding, ultrafiltration concentration, and affinity chromatography sequentially; wherein the denaturation dissolution uses a protein denaturing solution containing a denaturing agent, namely sodium dodecyl sulfate and / or CHAPS; and the dialysis refolding uses a protein dialysis refolding solution containing polyethylene glycol, arginine, and β-mercaptoethanol. This invention achieves efficient preparation of free recombinant canine CYP450 protein through the synergistic effect of the above purification routes, and the obtained protein possesses its native conformation and catalytic activity, solving the problem of the availability of high-quality canine CYP450 protein formulations from the source. Furthermore, the protein obtained by this method has the advantages of high purity and high yield.

Description

Technical Field

[0001] This invention belongs to the field of protein purification technology. Specifically, this invention relates to a method for purifying recombinant canine CYP450 protein, the recombinant canine CYP450 protein obtained by this method, a formulation containing the protein, a method for preparing the formulation, and its use in drug metabolism research. Background Technology

[0002] Dogs are the most critical non-rodent animal model for predicting the safety (especially cardiovascular toxicity) and metabolic characteristics of drugs in preclinical studies, and they play an irreplaceable role in drug development.

[0003] In canine preclinical studies, the CYP450 enzyme family is a core enzyme system for drug metabolism, and its activity directly affects the clearance rate, metabolic pathway, and potential toxicity risk of candidate compounds in vivo. Therefore, studying canine CYP450 enzymes is crucial for accurately interpreting drug metabolism data from canine preclinical studies, avoiding misjudgments of toxicity due to species differences, and effectively bridging to human metabolic characteristics.

[0004] High-purity canine-derived CYP450 enzymes have broad application value. First, they can mimic the metabolic process of drugs in dogs in vitro, allowing researchers to quickly eliminate poorly metabolized or toxic metabolites before animal testing, thus reducing unnecessary canine experiments and conforming to the 3R principle (reduction, recovery, and resilience). Second, high-purity canine-derived CYP450 enzymes can clearly distinguish the metabolic differences between canine and human CYP enzymes for the same substrate, helping to explain species-specific toxicities and avoiding data misinterpretation due to differences in metabolic characteristics. Furthermore, this enzyme can be used to optimize dosing regimens and guide toxicological experimental design, reducing the risk of failure in later clinical development.

[0005] Natural canine CYP450 enzymes need to be directly isolated and extracted from canine liver tissue. However, this method has many inherent drawbacks: it requires the sacrifice of experimental animals, which is costly and involves strict ethical review; the extraction process is complex and cumbersome; and more importantly, the purity and activity of the purified enzyme are difficult to meet the requirements of standardized research.

[0006] Heterologous expression using model microorganisms is an effective way to overcome the challenges of obtaining canine CYP450 enzymes. However, existing heterologous expression and purification systems for canine CYP450 enzymes still face significant technical bottlenecks. These enzymes are membrane proteins with highly hydrophobic molecular structures, making them prone to aggregation and precipitation after detachment from their native cell membrane environment, thus hindering the acquisition of free proteins that retain their native conformation and catalytic activity. This technical obstacle severely restricts the availability of high-quality canine CYP450 protein formulations, consequently affecting the accuracy and reliability of metabolic data interpretation in preclinical drug studies.

[0007] Therefore, there is an urgent need in this field to develop a purification method for canine CYP450 protein that can obtain high purity and has natural conformation and catalytic activity, in order to break through the current technical bottleneck and meet the urgent need for canine CYP450 protein formulations in the field of drug development. Summary of the Invention

[0008] To address the above-mentioned technical problems, the present invention aims to provide a purification method for obtaining high-purity recombinant canine CYP450 protein with its native conformation and catalytic activity. The inventors have discovered that by separating and extracting total membrane proteins from recombinant yeast fermentation broth, followed by gradient washing, denaturation and dissolution using sodium dodecyl sulfate (SDS) and / or 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS) as denaturing agents, dialysis and refolding with a protein dialysis and refolding solution containing polyethylene glycol, arginine, and β-mercaptoethanol, and then ultrafiltration concentration and affinity chromatography, the technical obstacle of obtaining free CYP450 protein with its native conformation and catalytic activity can be effectively overcome. Therefore, the present invention provides a purification method for recombinant canine CYP450 protein, the recombinant canine CYP450 protein obtained by this method, formulations containing the protein, and corresponding uses.

[0009] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0010] In a first aspect, the present invention provides a method for purifying recombinant canine CYP450 protein, comprising the following steps:

[0011] S01. Total membrane proteins were isolated and extracted from the fermentation broth of recombinant yeast expressing recombinant canine CYP450 protein;

[0012] S02. The total membrane proteins are subjected to gradient washing, denaturation and dissolution, dialysis refolding, ultrafiltration concentration and affinity chromatography in sequence;

[0013] The denaturing solution used for denaturation and dissolution contains a denaturing agent, which is SDS and / or CHAPS; and the protein dialysis refolding solution used for dialysis refolding contains polyethylene glycol, arginine, and β-mercaptoethanol.

[0014] This invention separates and extracts total membrane proteins from recombinant yeast fermentation broth. Gradient washing achieves precise separation of impurities from the target protein. A specific denaturing agent is then used to fully denature and unfold the highly hydrophobic membrane proteins. Combined with a specially formulated protein dialysis refolding solution, protein aggregation is effectively inhibited, and the target protein is promoted to refold correctly, restoring its native conformation and catalytic activity. Finally, ultrafiltration concentration and affinity chromatography yield high-purity target protein. The synergistic effect of each step in the above purification route enables the efficient preparation of free recombinant canine CYP450 protein, with the advantages of high purity, high yield, and preservation of native conformation and catalytic activity.

[0015] According to some embodiments of the present invention, the protein denaturing solution comprises: 0.3-2 g / 100 mL of denaturing agent, 300-500 mM NaCl, 10-20 vol% glycerol, and 0.5-1 mM β-mercaptoethanol; preferably, the protein denaturing solution comprises: 0.5-1 g / 100 mL of denaturing agent, 350-450 mM NaCl, 13-17 vol% glycerol, and 0.7-0.9 mM β-mercaptoethanol.

[0016] According to some embodiments of the present invention, the protein dialysis refolding solution comprises: 20-80 mM MOPS buffer, 1-5 g / 100 mL polyethylene glycol, 20-300 mM arginine, and 0.5-1 mM β-mercaptoethanol; preferably, the protein dialysis refolding solution comprises: 40-60 mM MOPS buffer, 2-4 g / 100 mL polyethylene glycol, 120-170 mM arginine, and 0.7-0.9 mM β-mercaptoethanol; preferably, the pH of the protein dialysis refolding solution is 6.5-7.9; more preferably, the MOPS buffer comprises: 20-80 mM Tris, 20-80 mM MOPS, 0.2-5 mM ethylenediaminetetraacetic acid (EDTA), and 0.05-2 g / 100 mL sodium dodecyl sulfate.

[0017] According to some embodiments of the present invention, the polyethylene glycol is selected from polyethylene glycols with a molecular weight of 6,000 to 10,000, preferably polyethylene glycol 6000.

[0018] According to some embodiments of the present invention, the protein dialysis refolding solution further comprises: 1-10 g / 100 mL of sucrose and / or 0.01-0.05 vol% of Tween 20.

[0019] According to some embodiments of the present invention, step S01 includes the following steps:

[0020] Step 1: Separate the solid and liquid components of the recombinant yeast fermentation broth, collect the bacterial sludge, and wash it at least once with buffer solution;

[0021] Step 2: Resuspend the washed bacterial sludge in a lysis solution containing lysozyme, add lysozyme to the obtained resuspension, break the cells to release the total intracellular protein, and collect the lysate;

[0022] Step 3: The lysate is subjected to ultracentrifugation and differential centrifugation in sequence to collect total membrane proteins.

[0023] Preferably, the recombinant yeast is selected from one or more of Saccharomyces cerevisiae, Pichia pastoris, Yersinia lipolytica, and Hansenula anomala, with Pichia pastoris being the most preferred.

[0024] Preferably, in step one, the buffer solution is a phosphate buffer solution; more preferably, the pH of the phosphate buffer solution is 7.2-7.6; more preferably, the phosphate buffer solution comprises: 6-10 g / L sodium chloride, 0.1-0.5 g / L potassium chloride, 1-2 g / L disodium hydrogen phosphate, and 0.1-0.5 g / L potassium dihydrogen phosphate.

[0025] Preferably, in step two, the pH of the lysis solution is 7.2-7.6.

[0026] Preferably, in step two, the lysis solution comprises: 20-80 mM MOPS buffer, 5-20 vol% glycerol, 1-5 vol‰ protease inhibitor, 2-8 vol‰ lysozyme, 0.5-1 mM β-mercaptoethanol, and 50-100 U / 100 mL totipotent nuclease.

[0027] Preferably, in step two, the amount of the lysozyme added is 0.5-2 volume.

[0028] Preferably, in step two, the cell disruption includes: a disruption temperature of 2-10℃; a disruption time of 15-60 min; and a disruption pressure of 1200-1500 bar.

[0029] According to some specific embodiments of the present invention, step S01 includes the following steps:

[0030] Step 1: Separate the solid and liquid components of the recombinant yeast fermentation broth and collect the bacterial sludge; resuspend the bacterial sludge in phosphate buffer solution, and then perform solid-liquid separation again to collect the precipitate and obtain the washed bacterial sludge.

[0031] Step 2: Resuspend the washed bacterial sludge in a lysis solution containing lysing enzymes, adjust the concentration of the obtained bacterial suspension to OD600=50, add 0.5-2% by volume of lysing enzymes, stir evenly, and then use a homogenizer to break the cells at 2-10℃ and 1200-1500 bar for 15-60 minutes to release the total intracellular protein and collect the lysate.

[0032] Step 3: The lysate is subjected to ultracentrifugation to collect the precipitate. Cell debris is then removed from the precipitate by differential centrifugation to collect the total membrane protein.

[0033] According to some embodiments of the present invention, in step S02, the gradient washing includes: performing gradient washing on the total membrane proteins using a membrane protein washing solution with progressively increasing detergent concentration until no protein is detected in the supernatant, and collecting the precipitate after washing.

[0034] This invention employs a gradient washing method using membrane protein washing solutions containing different concentrations of detergent to wash total membrane proteins. By utilizing the differences in solubility of different proteins in detergent, as the detergent concentration gradually increases, impurity proteins are dissolved and removed sequentially, thereby achieving precise separation of impurity proteins from recombinant canine CYP450 protein and effectively reducing the difficulty of subsequent purification.

[0035] Preferably, in the gradient washing, each gradient is incubated at 2-10°C for 10-60 min by rotation and then centrifuged; when no protein is detected in the supernatant, the precipitate is washed with a membrane protein washing solution without detergent and then centrifuged.

[0036] Preferably, the detergent is urea and / or sodium cholate; more preferably, the membrane protein washing solution comprises: 40-60 mM Tris-HCl, 1-5 mM EDTA, 0.5-2 vol% Tween 100, 0.2-0.8 M NaCl, 2-8 M urea and / or 1-5 g / 100 mL sodium cholate; more preferably, the difference in urea concentration between two adjacent washes is 1-3 M; more preferably, the difference in sodium cholate concentration between two adjacent washes is 1-3 g / 100 mL.

[0037] According to some embodiments of the present invention, in step S02, the denaturation and dissolution includes: contacting the precipitate after gradient washing with the protein denaturation solution to dissolve the target protein, separating and removing insoluble matter, and collecting the solution containing the denatured target protein.

[0038] Preferably, the process of dissolving the target protein includes: rotating and incubating at 2-10°C for 1-3 hours.

[0039] Preferably, the weight ratio of the precipitate after gradient washing to the volume ratio of the protein denaturation solution is 1:5-15, more preferably 1:8-12.

[0040] According to some embodiments of the present invention, in step S02, the dialysis refolding includes: placing a solution containing the denatured target protein in a dialysis bag, and performing dialysis in the protein dialysis refolding solution to refold the target protein.

[0041] Preferably, the molecular weight cutoff of the dialysis bag is 11-13 kDa.

[0042] Preferably, the dialysis is performed at 2-10°C for 14 hours or more, and more preferably for 14-20 hours.

[0043] According to some embodiments of the present invention, in step S02, the ultrafiltration concentration includes: concentrating the dialyzed and refolded protein solution using an ultrafiltration tube.

[0044] Preferably, the ultrafiltration tube has a molecular weight cutoff of 28-32 kDa.

[0045] Preferably, the concentration is carried out at 2-10°C with a centrifugal force of 3000-3500×g.

[0046] Preferably, the concentration of the concentrated protein is 5-10 mg / mL.

[0047] According to some embodiments of the present invention, in step S02, the affinity chromatography includes: loading the concentrated protein solution onto an affinity chromatography column, performing column equilibration, loading, eluting impurities and eluting the target protein in sequence, and collecting the chromatographic eluent of the target protein.

[0048] Preferably, the C-terminus of the recombinant canine CYP450 protein is fused with a 6×His tag, and the affinity chromatography is nickel affinity chromatography; more preferably, the working pH of the nickel affinity chromatography column is 6.0 to 8.0.

[0049] This invention utilizes the specific affinity adsorption between a 6×His tag and nickel packing material. Impurities are removed by gradient elution with a low concentration of imidazole, while the target protein is competitively eluted with a high concentration of imidazole. High-purity target protein can be obtained with only one nickel affinity chromatography step, resulting in high purification efficiency, a simple process, and suitability for scale-up production.

[0050] Preferably, the elution of impurities is performed using a buffer solution containing 20-80 mM imidazole.

[0051] Preferably, the target protein is eluted using a buffer solution containing 150-500 mM imidazole.

[0052] According to some specific embodiments of the present invention, step S02 includes the following steps:

[0053] (1) Membrane protein gradient washing: The total membrane proteins were washed with a membrane protein washing solution with progressively increasing detergent concentration. Each gradient was incubated at 2-10℃ for 10-60 min and then centrifuged. The washing continued until no protein was detected in the supernatant. The precipitate was then washed with a membrane protein washing solution without detergent and collected by centrifugation.

[0054] (2) Denaturation and dissolution of target protein: The precipitate after gradient washing is contacted with the protein denaturation solution and incubated at 2-10℃ for 1-3 hours to fully dissolve the target protein; the obtained solution is centrifuged to remove insoluble matter and the supernatant is collected, which is the solution containing the denatured target protein.

[0055] (3) Dialysis refolding: The solution containing the denatured target protein is placed in an activated dialysis bag with a molecular weight cutoff of 11-13 kDa, and the protein is dialyzed in the protein dialysis refolding solution at 2-10°C for 14 hours or more to refold the target protein. The protein solution in the bag is then collected.

[0056] (4) Ultrafiltration concentration: The protein solution after dialysis and refolding is concentrated by ultrafiltration at 2-10℃ with a centrifugal force of 3000-3500×g using an ultrafiltration tube with a molecular weight cutoff of 28-32kDa, until the protein concentration is 5-10mg / mL.

[0057] (5) Nickel affinity chromatography purification: The concentrated protein solution is loaded onto a nickel affinity chromatography column, and the column is equilibrated, loaded, eluted with impurities and eluted with the target protein in sequence; impurities are eluted with a buffer containing 20-80 mM imidazole, and the target protein is eluted with a buffer containing 150-500 mM imidazole. The chromatographic eluent of the target protein is collected.

[0058] In a second aspect, the present invention provides a recombinant canine CYP450 protein, which is obtained by the purification method described in the first aspect of the present invention.

[0059] Preferably, the recombinant canine CYP450 protein is selected from one or more of canine CYP1A1 (KP340901.1), canine CYP1A2 (NM_001008720.1), canine CYP2B11 (NM_001006652.1), canine CYP2C21 (NM_001197044.1), canine CYP2C41 (NM_001003334.1), canine CYP2D15 (NM_001003333.1), canine CYP2E1 (NM_001003339.1), canine CYP3A12 (NM_001003340.1), and canine CYP3A26 (NM_001003338.1).

[0060] Thirdly, the present invention provides a recombinant canine CYP450 protein formulation comprising the recombinant canine CYP450 protein according to the second aspect of the present invention or the recombinant canine CYP450 protein obtained by the purification method according to the first aspect of the present invention.

[0061] Fourthly, the present invention provides a method for preparing a recombinant canine CYP450 protein formulation, wherein the preparation method includes: ultrafiltration concentration, dialysis desalting, and cryopreservation of the chromatographic eluent of the target protein obtained by the purification method according to the first aspect of the present invention.

[0062] This invention utilizes affinity chromatography eluent to concentrate it through ultrafiltration, followed by dialysis to remove salts, effectively removing high concentrations of imidazole. After adding a cryoprotectant, the protein is rapidly frozen for preservation, thereby maintaining the long-term stability and biological activity of the target protein.

[0063] According to some embodiments of the present invention, the preparation method includes the following steps: ultrafiltration and concentration of the chromatographic eluent of the target protein to a protein concentration of 1-5 mg / mL, then placing it in a dialysis bag, dialyzing it in a dialysis solution to remove salt, then adding glycerol at a final concentration of 10-30% by volume as a cryoprotectant, and storing it at -70 to -90°C after quick freezing in liquid nitrogen.

[0064] Preferably, the molecular weight cutoff of the dialysis bag is 11-13 kDa.

[0065] Preferably, the dialysis desalination is performed at 2-10°C for 14 hours or more, more preferably 14-20 hours.

[0066] According to some specific embodiments of the present invention, the preparation method includes the following specific steps:

[0067] (1) Ultrafiltration concentration: The chromatographic eluent of the target protein was subjected to SDS-PAGE detection. After Coomassie brilliant blue staining and gray scale comparison, the eluent with a purity of more than 90% was selected. The selected eluent was concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 28-32kDa at 2-10℃ with a centrifugal force of 3000-3500×g to concentrate the protein concentration to 1-5mg / mL.

[0068] (2) Desalting and cryopreservation: The concentrated protein solution is placed in an activated dialysis bag with a molecular weight cutoff of 11-13 kDa and dialyzed in a protein dialysis refolding solution at 2-10°C for 14 hours or more to remove high concentrations of imidazole; after dialysis, glycerol is added to a final concentration of 10-30% by volume as a cryoprotectant, and after quick freezing in liquid nitrogen, it is stored at -70°C to -90°C.

[0069] Fifthly, the present invention provides the use of the recombinant canine CYP450 protein according to the second aspect of the present invention or the recombinant canine CYP450 protein formulation according to the third aspect of the present invention in the preparation of formulations for drug metabolism studies.

[0070] The present invention has at least the following beneficial effects:

[0071] 1. This invention provides a method for purifying recombinant canine CYP450 protein. The method first separates and extracts total membrane proteins from recombinant yeast fermentation broth, ensuring the initial source of the target protein. Gradient washing of the total membrane proteins achieves precise separation of impurities from the target protein, laying the foundation for subsequent purification. Then, a specific denaturing agent is used to fully denature and unfold the highly hydrophobic membrane proteins, improving their solubility. Refolding is then performed using a protein dialysis refolding solution of a specific composition, effectively inhibiting protein aggregation and allowing the denatured target protein to refold correctly, restoring its native conformation and catalytic activity. Finally, high-purity target protein is obtained through ultrafiltration concentration and affinity chromatography. This invention, through the synergistic effect of the above-mentioned specific purification route, achieves for the first time the efficient preparation of bioactive free recombinant canine CYP450 protein, solving the problem of the availability of high-quality canine CYP450 protein formulations from the source.

[0072] 2. The purification method provided by this invention has the advantages of high purity and high yield. Specifically, the purity of the recombinant canine CYP450 protein obtained using the method of this invention reaches 92% or higher, and the pure product yield per liter of fermentation broth can reach approximately 47 mg or more.

[0073] 3. The purification method provided by this invention has advantages such as high timeliness, simple operation, and strong versatility. Specifically, the entire process of obtaining high-purity recombinant canine CYP450 protein from fermentation broth can be controlled within 48 hours, which is beneficial for preserving the native conformation and catalytic activity of the recombinant canine CYP450 protein. Moreover, the purification method of this invention only requires one nickel affinity chromatography step to achieve the required purity, the process is simple, and it is suitable for scale-up production. Furthermore, this method can be extended to the purification of a range of recombinant canine CYP450 proteins.

[0074] 4. The high-purity canine CYP450 protein obtained using the purification method provided in this invention has significant application value. Specifically, this protein possesses the correct native conformation and catalytic activity, allowing it to simulate the metabolic process of drugs in dogs in vitro. This enables the rapid elimination of candidate compounds with poor metabolism or that easily produce toxic metabolites before animal experiments, thereby reducing unnecessary canine studies. Simultaneously, this protein helps distinguish the metabolic differences between canine and human CYP proteins for the same substrate, avoiding data misinterpretation due to different metabolic characteristics. Furthermore, it can be used to optimize dosing regimens and guide toxicology experiment design, effectively bridging preclinical and clinical data, reducing the risk of later-stage development failure, and providing a reliable tool for interpreting metabolic data in preclinical drug research.

[0075] In summary, this invention has achieved high-purity preparation of free recombinant canine CYP450 protein with the correct natural conformation and catalytic activity. Significant progress has been made in terms of technical feasibility, product quality, process efficiency, and application, laying a technical foundation for the standardized supply of canine CYP450 protein formulations in preclinical drug research. Attached Figure Description

[0076] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0077] Figure 1 This shows an SDS-PAGE electrophoresis image of the purified target protein from Example 1 of the present invention. Wherein, M: protein marker; lane 1: purified product.

[0078] Figure 2 The images show the LC-MS / MS total ion chromatogram and characteristic product ion mass spectrum of the recombinant canine CYP3A12 protein-catalyzed testosterone 6β-hydroxylation reaction in Example 1 of this invention; wherein, Figure 2 A is the negative control group substrate testosterone ion chromatogram; Figure 2 B is the testosterone ion chromatogram after the substrate has been catalyzed by the enzyme for 30 min; Figure 2 C is the ion chromatogram of 6β-hydroxytestosterone after enzyme catalysis of the substrate for 30 min;

[0079] Figure 3 The images show the LC-MS / MS total ion chromatogram and characteristic product ion mass spectrum of the testosterone 6β-hydroxylation reaction catalyzed by recombinant canine CYP3A26 protein in Example 2 of this invention; wherein, Figure 3 A is the negative control group substrate testosterone ion chromatogram; Figure 3 B is the testosterone ion chromatogram after the substrate has been catalyzed by the enzyme for 30 min; Figure 3 C is the ion chromatogram of 6β-hydroxytestosterone after enzyme catalysis for 30 min. Detailed Implementation

[0080] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0081] Unless otherwise specified, the methods or conditions used in the following examples were performed according to conventional methods disclosed in the art. Unless otherwise stated, all reagents or instruments used in the following examples are commercially available products.

[0082] In this invention, the term "cytochrome P450 (CYP450)" refers to a superfamily of monooxygenases with iron porphyrin (heme) as a cofactor. It is named for the characteristic absorption peak at a wavelength of 450 nm after the reduced state binds to carbon monoxide. It is widely involved in the oxidative metabolism of endogenous and exogenous substances.

[0083] In this invention, the term "recombinant canine CYP450 protein" refers to canine cytochrome P450 protein obtained by expressing it in a heterologous host using recombinant DNA technology.

[0084] In this invention, the term "total membrane protein" refers to the collection of all proteins in a cell that are associated with biological membranes (including cell membranes and various organelle membranes); specifically, in this invention, it refers to the membrane protein components obtained by separating and precipitating from broken yeast cells through differential centrifugation or ultracentrifugation.

[0085] In this invention, the term "6×His tag" refers to a short peptide sequence consisting of 6 consecutive histidine residues, which specifically chelates with transition metal ions such as nickel ions through the imidazole ring on the side chain of the histidine residues, thereby achieving immobilized metal ion affinity chromatography purification of the target protein.

[0086] In this invention, the term "Pichia pastoris" refers to the methanol-nutritive yeast Pichia pastoris (now taxonomically known as Komagataella phaffi), a system commonly used for the eukaryotic expression of recombinant proteins.

[0087] In this invention, the term "gradient washing" refers to the operation of treating membrane protein precipitates stepwise using washing solutions containing different concentrations of detergent; wherein the detergent concentration is gradually increased, and proteins with different solubilities are dissolved and removed sequentially at different detergent concentrations, thereby achieving the separation of target membrane proteins from impurity proteins.

[0088] In this invention, the term "denaturing dissolution" refers to the process of using a denaturing agent to disrupt the native conformation of a protein, causing its polypeptide chains to unfold and dissolve in a solution.

[0089] In this invention, the term "dialysis refolding" refers to the process of placing a denatured protein solution in a dialysis bag and slowly dialyzing it with a dialysis refolding solution to gradually remove the denaturing agent, thereby causing the denatured protein polypeptide chains to refold into a three-dimensional conformation with natural biological activity.

[0090] In this invention, the term "ultrafiltration concentration" refers to the operation of using an ultrafiltration membrane with a specific molecular weight cutoff to allow water and small molecule solutes to pass through the membrane pores under centrifugal force or pressure, while proteins larger than the molecular weight cutoff are retained and enriched.

[0091] In this invention, the term "affinity chromatography" refers to a chromatography technique that utilizes the reversible interaction between a biomacromolecule and its specific ligand to achieve the separation and purification of a target molecule; specifically, in this invention, it refers to nickel affinity chromatography that uses the specific affinity adsorption between a 6×His tag and nickel ions to capture the target protein.

[0092] In this invention, the term "protease inhibitor" refers to a class of small molecule compounds or peptides that can inhibit protease activity and prevent target proteins from being degraded by endogenous proteases.

[0093] In this invention, the term "cell wall lysing enzyme" refers to a hydrolytic enzyme that can hydrolyze components such as β-1,3-glucan, β-1,6-glucan, and chitin in the yeast cell wall, thereby degrading the yeast cell wall and assisting in the release of cell contents.

[0094] In this invention, the term "totipotent nuclease" refers to a nuclease that can non-specifically degrade DNA and RNA, and is used to reduce the viscosity of the lysate after cell disruption.

[0095] In this invention, the term "cryopreservation protectant" refers to a substance that can protect proteins from ice crystal damage and conformational changes during cryopreservation; specifically, in this invention, the cryopreservation protectant is glycerol.

[0096] In this invention, the term "membrane protein" refers to a protein that can bind to or integrate into the cell membrane or organelle membrane; canine CYP450 enzyme belongs to the category of integrated membrane proteins.

[0097] The source and composition of the MOPS buffer used in the following examples are as follows:

[0098] MOPS buffer: purchased from Solarbio 10×MOPS, catalog number: 2500080002, its 1× working concentration contains: 50 mM Tris, 50 mM MOPS, 0.1 g / 100 mL SDS, 1 mM EDTA, pH 7.5-7.9.

[0099] Example 1

[0100] This embodiment provides a purification method for recombinant canine CYP3A12 protein (amino acid sequence corresponding to GenBank accession number NM_001003340.1, with a 6×His tag at the C-terminus), specifically including the following steps:

[0101] I. Extraction of total membrane proteins from fermentation broth

[0102] 1. Preparation of Pichia pastoris fermentation broth

[0103] The canine-derived CYP3A12 heterologous expression gene was transformed into Pichia pastoris competent cells, and recombinant Pichia pastoris strains were obtained through screening. These recombinant Pichia pastoris strains were inoculated into seed culture medium with glycerol as the carbon source and cultured at 28℃ and 180-220 rpm with shaking for 48 h. After removing the seed culture medium, it was replaced with induction culture medium with methanol as the carbon source, and cultured under the same conditions for another 72 h to induce recombinant protein expression. The fermentation broth was collected for subsequent extraction of total membrane proteins.

[0104] The competent Pichia pastoris cells were purchased from Yuanye Biotechnology (catalog number: S22725-100μL×10). The seed culture medium consisted of: 20g / L peptone, 10g / L yeast extract, 3.4g / L YNB, 10g / L ammonium sulfate, 1v% glycerol, and 10% (v / v) 1M potassium phosphate buffer (pH 6.0). The induction culture medium consisted of: 20g / L peptone, 10g / L yeast extract, 3.4g / L YNB, 10g / L ammonium sulfate, 1v% methanol, and 10% (v / v) 1M potassium phosphate buffer (pH 6.0).

[0105] 2. Separation and washing of mycelium sludge

[0106] Take 1 L of the Pichia pastoris fermentation broth expressing recombinant canine CYP3A12 protein prepared above, centrifuge at 8000×g for 15 min at 4℃, collect the bacterial sludge, and discard the supernatant. Resuspend the bacterial sludge in pre-cooled phosphate buffer solution (8.0 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, diluted to 1 L of purified water, pH 7.4), and centrifuge again at 8000×g for 15 min at 4℃, collect the precipitate, and obtain the Pichia pastoris bacterial sludge expressing the target protein.

[0107] 3. Extraction of total membrane proteins

[0108] The bacterial sludge was resuspended in a pre-cooled lysis solution, the OD600 was adjusted to 50, 1% (v / v) of lysozyme was added, and the mixture was stirred thoroughly. The mixture was then homogenized using a high-pressure homogenizer at 4°C and 1300 bar for 30 min, repeated three times to release total intracellular protein. The lysis solution contained: 1×MOPS buffer, 10% (v / v) glycerol, 2% (v / v) protease inhibitor, 5% (v / v) lysozyme, 0.8 mM β-mercaptoethanol, and 80 U / 100 mL totipotent nuclease.

[0109] The lysate was centrifuged at 100,000 × g for 60 min at 4 °C, and the precipitate was collected. The precipitate was resuspended in pre-cooled lysis buffer (without cell wall lysing enzymes), centrifuged at 6,000 × g for 30 min at 4 °C to remove cell wall polysaccharides, and the supernatant was collected. The supernatant was then centrifuged at 100,000 × g for 60 min at 4 °C, and the precipitate was collected to obtain total membrane proteins.

[0110] II. Purification of the target protein

[0111] 1. Membrane protein gradient washing

[0112] The total membrane protein precipitate obtained in the above steps was suspended in membrane protein washing solution A (containing: 50 mM Tris-HCl pH 8.8, 2 mM EDTA, 1 v / v Tween 100, 0.5 M NaCl and 2 M urea), and incubated at 4 °C by rotation for 30 min. Then, it was centrifuged at 20000 × g at 4 °C for 30 min and the supernatant was discarded.

[0113] The obtained precipitate was washed once more with membrane protein washing solution B (containing: 50 mM Tris-HCl pH 8.8, 2 mM EDTA, 1 v / v Tween 100, 0.5 M NaCl and 4 M urea).

[0114] Finally, wash once with urea-free membrane protein washing solution C (containing: 50mM Tris-HCl pH 8.8, 2mM EDTA, 1 v / v Tween 100 and 0.5M NaCl), and collect the precipitate by centrifugation.

[0115] 2. Target protein denaturation and dissolution

[0116] The washed precipitate was added to a protein denaturing solution at a ratio of 1:10 (w / v). The mixture was incubated at 4°C for 2 hours by rotation to ensure complete dissolution of the target protein. The resulting solution was then centrifuged at 100,000 × g for 45 minutes at 4°C, and the supernatant was collected as the denatured target protein solution. The protein denaturing solution contained: 0.5 g / 100 mL SDS, 400 mM NaCl, 15 v / v glycerol, and 0.8 mM β-mercaptoethanol.

[0117] 3. Dialysis refolding

[0118] The denatured target protein solution was placed into an activated dialysis bag (molecular weight cutoff 12 kDa), with the protein dialysis refolding solution outside the dialysis bag. Dialysis was performed at 4°C with magnetic stirring for 16 hours, with the protein dialysis refolding solution replaced every 8 hours. After dialysis refolding, the protein solution inside the bag was collected. The protein dialysis refolding solution contained: 50 mM MOPS buffer, 3 g / 100 mL PEG6000, 150 mM arginine, and 0.8 mM β-mercaptoethanol, pH 7.4.

[0119] 4. Ultrafiltration Concentration

[0120] The dialyzed and refolded protein solution was transferred to a Millipore ultrafiltration tube with a molecular weight cutoff of 30 kDa and concentrated at 3500 × g at 4 °C to a protein concentration of approximately 8 mg / mL (determined by the BCA method), yielding a concentrated dialyzed and refolded protein solution (approximately 15 mL).

[0121] 5. Nickel affinity chromatography purification

[0122] Purification was performed using a 5 mL HisTrap FF pre-packed column (Cytiva) in conjunction with the AKTA pure system. All buffers were filtered through a 0.45 μm filter membrane.

[0123] Column equilibration: Equilibrate the chromatography column with 5 column volumes of equilibration buffer (containing: 1×MOPS buffer, 300mM NaCl, 20% glycerol, 20mM imidazole, pH 7.4).

[0124] Sample loading: Load the above concentrated dialysis refolded protein solution onto the chromatography column at a flow rate of 1 mL / min.

[0125] Low-concentration imidazole washing: Wash the chromatography column with 10 column volumes of washing buffer (containing: 1×MOPS buffer, 300mM NaCl, 20% glycerol, 50mM imidazole, pH 7.4) until the UV absorption peak drops to baseline to remove contaminating proteins.

[0126] High-concentration imidazole elution: Elute the target protein with 5 column volumes of elution buffer (containing: 1×MOPS buffer, 300mM NaCl, 20% glycerol, 250mM imidazole, pH 7.4) and collect the elution peak.

[0127] III. Preparation of Target Protein Formulation

[0128] 1. Ultrafiltration Concentration

[0129] The chromatographic eluent of the target protein was analyzed by SDS-PAGE. After Coomassie brilliant blue staining and grayscale comparison, the eluent with a purity of over 90% was selected and concentrated to a protein concentration of approximately 3 mg / mL by centrifugation at 3500 × g at 4°C using an ultrafiltration tube with a molecular weight cutoff of 30 kDa.

[0130] 2. Desalination and preparation

[0131] The concentrated protein solution was placed into activated dialysis bags (molecular weight cutoff 12 kDa) and dialyzed overnight (14 h) at 4°C with protein dialysis solution (containing: 1×MOPS buffer, 3% PEG6000, 150 mM arginine, 0.8 mM β-mercaptoethanol, 10% glycerol) to remove high concentrations of imidazole. After dialysis, sterile glycerol was added to a final concentration of 20% by volume, mixed well, aliquoted, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0132] IV. Experimental Results

[0133] Using the above method, approximately 49 mg of pure recombinant canine CYP3A12 protein was finally obtained from 1 L of fermentation broth, which translates to a yield of approximately 49 mg per liter of fermentation broth.

[0134] SDS-PAGE analysis (see) Figure 1 Lane M: protein molecular weight standard; Lane 1: purified product), the target protein band was located at approximately 55 kDa, consistent with the theoretical molecular weight. ImageJ software grayscale scanning analysis showed the target protein purity to be approximately 92%. The final protein concentration, determined by BCA method, was 2.8 mg / mL.

[0135] Example 2

[0136] This embodiment provides a purification method for recombinant canine CYP3A26 protein (amino acid sequence corresponding to GenBank accession number NM_001003338.1, with a 6×His tag at the C-terminus). This embodiment follows the same core route as Example 1, but some specific details have been adjusted according to the characteristics of the target protein. The specific steps are as follows:

[0137] I. Extraction of total membrane proteins from fermentation broth

[0138] 1. Preparation of Pichia pastoris fermentation broth

[0139] Prepared according to the method of Example 1, the difference from Example 1 is that the canine CYP3A12 heterologous expression gene is replaced with the canine CYP3A26 heterologous expression gene.

[0140] 2. Separation and washing of mycelium sludge

[0141] Same as Example 1.

[0142] 3. Extraction of total membrane proteins

[0143] Same as Example 1.

[0144] II. Purification of the target protein

[0145] 1. Membrane protein gradient washing

[0146] The total membrane protein precipitate obtained in the above steps was suspended in membrane protein washing solution A (containing: 50 mM Tris-HCl pH 8.8, 2 mM EDTA, 1 v / v Tween 100, 1 g / 100 mL sodium cholate, 0.5 M NaCl), and incubated at 4 °C with rotation for 30 min. Then, it was centrifuged at 20000 × g at 4 °C for 30 min, and the supernatant was discarded.

[0147] The obtained precipitate was washed once more with membrane protein washing solution B (containing: 50mM Tris-HCl pH 8.8, 2mM EDTA, 1% Tween 100, 2g / 100mL sodium cholate, 0.5M NaCl), and the supernatant was discarded.

[0148] In view of the characteristics of recombinant canine CYP3A26 protein, this embodiment adds a washing step, the specific steps of which are as follows: the obtained precipitate is washed again with membrane protein washing solution C (containing: 50mM Tris-HCl pH 8.8, 2mM EDTA, 1 volume% Tween 100, 5g / 100mL sodium cholate, 0.5M NaCl), and the supernatant is discarded.

[0149] The obtained precipitate was washed once with a detergent-free membrane protein washing solution D (50mM Tris-HCl pH 8.8, 2mM EDTA, 1 v / v Tween 100, 0.5M NaCl), and the final precipitate was collected by centrifugation.

[0150] 2. Target protein denaturation and dissolution

[0151] The washed precipitate was added to a protein denaturing solution at a ratio of 1:10 (w / v). The mixture was incubated at 4°C for 2.5 h (CHAPS dissolves slightly slower than SDS, so the incubation time was appropriately extended) to ensure complete dissolution of the target protein. The resulting solution was then centrifuged at 100,000 × g for 45 min at 4°C, and the supernatant was collected as the denatured target protein solution. The protein denaturing solution contained: 1 g / 100 mL CHAPS, 400 mM NaCl, 15 v / v glycerol, and 0.8 mM β-mercaptoethanol.

[0152] 3. Dialysis refolding

[0153] Same as Example 1.

[0154] 4. Ultrafiltration Concentration

[0155] Same as Example 1.

[0156] 5. Nickel affinity chromatography purification

[0157] Same as Example 1.

[0158] III. Preparation of Target Protein Formulation

[0159] 1. Ultrafiltration Concentration

[0160] Same as Example 1.

[0161] 2. Desalination and preparation

[0162] The concentrated protein solution was placed into activated dialysis bags (molecular weight cutoff 12 kDa) and dialyzed overnight (14 h) at 4°C with protein dialysis solution (containing: 1×MOPS buffer, 3% PEG6000, 150 mM arginine, 0.8 mM β-mercaptoethanol, 5 g / 100 mL sucrose, and 0.02 g / 100 mL Tween 20) to remove high concentrations of imidazole. After dialysis, sterile glycerol was added to a final concentration of 20%, mixed well, aliquoted, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0163] IV. Experimental Results

[0164] Using the above method, approximately 47.6 mg of pure recombinant canine CYP3A26 protein was finally obtained from 1 L of fermentation broth, which translates to a yield of approximately 47.6 mg per liter of fermentation broth.

[0165] SDS-PAGE analysis showed that the target protein band was located at approximately 55 kDa, consistent with the theoretical molecular weight; ImageJ software grayscale scanning analysis showed that the purity of the target protein was approximately 95.5%; the final protein concentration determined by BCA method was 2.7 mg / mL.

[0166] Example 3

[0167] The recombinant canine CYP3A12 protein was purified according to the method in Example 1, except that the denaturing agent was 0.5 g / 100 mL CHAPS.

[0168] Using the above method, approximately 56 mg of pure recombinant canine CYP3A12 protein was finally obtained from 1 L of fermentation broth, which translates to a yield of approximately 56 mg per liter of fermentation broth.

[0169] SDS-PAGE analysis and ImageJ software grayscale scanning analysis showed that the purity of the target protein was approximately 96%. The final protein concentration was determined to be 3 mg / mL by BCA method.

[0170] Activity verification of recombinant canine CYP protein (LC-MS / MS mass spectrometry)

[0171] I. Construction of the enzyme activity assay system

[0172] The catalytic cycle of CYP proteins requires electrons from NADPH. The monooxygenase reaction system constructed in this invention is as follows: 5 μL of substrate (dissolved in 40% vol% methanol, final concentration 5 μM), 80 μL of recombinant canine CYP protein, 80 μL of NADPH solution, 10 μL of cytochrome B5 enzyme, and 100 mM PBS solution to a final volume of 200 μL.

[0173] After incubating the reaction at 37°C for 30 min, 600 μL of ice-cold methanol was added to terminate the reaction. The mixture was then centrifuged at 12000×g for 10 min, and the supernatant was collected for LC-MS / MS analysis.

[0174] II. Selection of Characteristic Substrates

[0175] The characteristic substrates and characteristic metabolites corresponding to each canine CYP subtype are shown in Table 1.

[0176]

[0177] III. LC-MS / MS Detection

[0178] LC-MS / MS detection was performed using an ultra-high performance liquid chromatography-quadrupole electrostatic track trap high-resolution mass spectrometer (Thermo Scientific Q Exactive) equipped with a heated electrospray ionization source (HESI), and the following parameters were used for detection:

[0179] Chromatographic conditions: A Waters ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, 3.5 μm) was used; mobile phase A was 0.1 vol% formic acid aqueous solution, and mobile phase B was acetonitrile; the gradient elution program was: 0–5 min, 5–95 vol% B; 5–7 min, 95 vol% B; 7–7.1 min, 9–5 vol% B; 7.1–9 min, 5 vol% B; flow rate 0.3 mL / min; column temperature 30℃.

[0180] Mass spectrometry conditions: Heated electrospray ionization source (HESI) was used, with positive and negative ion switching scanning modes; spray voltage (+ / -): 3500 / 2500V, sheath gas pressure: 35Arb, auxiliary gas pressure: 10Arb, purge gas pressure: 0Arb, ion transfer tube temperature: 350℃, auxiliary gas heating temperature: 325℃.

[0181] IV. Mass Spectrometry Validation Results

[0182] The reaction systems catalyzed by canine CYP3A12 and canine CYP3A26 proteins for the 6β-hydroxylation of testosterone were used as validation models to evaluate the catalytic activity of the recombinant canine CYP450 protein prepared in this invention. The results are as follows: Figure 2 and Figure 3 As shown.

[0183] The results show that after metabolism catalyzed by canine CYP3A12 and canine CYP3A26 proteins, the peak area of ​​the substrate chromatogram decreased significantly, and a specific product peak appeared at a retention time of 4.45 min, which was absent in the blank control group. Mass spectrometry analysis indicated that the product had a quasi-molecular ion peak [M+H]. + The value was 305.2, which is consistent with the theoretical molecular weight of 6β-hydroxytestosterone; its secondary mass spectrometry can detect two sets of characteristic fragment ions at m / z 269.133 and m / z 108.967, which is consistent with the mass spectrometric fragmentation pattern of 6β-hydroxytestosterone standards.

[0184] The above mass spectrometry results confirm that the recombinant canine CYP450 protein prepared in this invention has the correct native conformation and catalytic activity, and can specifically catalyze the oxidative metabolic reaction of the characteristic substrate to generate the expected characteristic metabolites.

[0185] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or similar implementation schemes obtained by those skilled in the art by making some modifications or alterations to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. A method for purifying recombinant canine CYP450 protein, wherein, The method includes the following steps: S01. Total membrane proteins were isolated and extracted from the fermentation broth of recombinant yeast expressing recombinant canine CYP450 protein; S02. The total membrane proteins are subjected to gradient washing, denaturation and dissolution, dialysis refolding, ultrafiltration concentration and affinity chromatography in sequence; The denaturing solution used for denaturation and dissolution contains a denaturing agent, which is sodium dodecyl sulfate and / or CHAPS; and the protein dialysis refolding solution used for dialysis refolding contains polyethylene glycol, arginine and β-mercaptoethanol.

2. The purification method according to claim 1, wherein, The protein denaturing solution comprises: 0.3-2 g / 100 mL of denaturing agent, 300-500 mM NaCl, 10-20 vol% glycerol, and 0.5-1 mM β-mercaptoethanol; Preferably, the protein denaturing solution comprises: 0.5-1 g / 100 mL of denaturing agent, 350-450 mM NaCl, 13-17 vol% glycerol, and 0.7-0.9 mM β-mercaptoethanol; Preferably, the protein dialysis refolding solution comprises: 20-80 mM MOPS buffer, 1-5 g / 100 mL polyethylene glycol, 20-300 mM arginine, and 0.5-1 mM β-mercaptoethanol; More preferably, the protein dialysis refolding solution comprises: 40-60 mM MOPS buffer, 2-4 g / 100 mL polyethylene glycol, 120-170 mM arginine, and 0.7-0.9 mM β-mercaptoethanol; Preferably, the polyethylene glycol is selected from polyethylene glycol with a molecular weight of 6,000 to 10,000, and more preferably polyethylene glycol 6000; Preferably, the protein dialysis refolding solution further comprises: 1-10 g / 100 mL of sucrose and / or 0.01-0.05 g / 100 mL of Tween 20.

3. The purification method according to claim 1 or 2, wherein, Step S01 includes the following steps: Step 1: Separate the solid and liquid components of the recombinant yeast fermentation broth, collect the bacterial sludge, and wash it at least once with buffer solution; Step 2: Resuspend the washed bacterial sludge in a lysis solution containing lysozyme, add lysozyme to the obtained resuspension, break the cells to release the total intracellular protein, and collect the lysate; Step 3: The lysate is subjected to ultracentrifugation and differential centrifugation in sequence to collect total membrane proteins; Preferably, in step one, the buffer solution is a phosphate buffer solution with a pH of 7.2-7.6; Preferably, in step two, the lysis solution comprises: 20-80 mM MOPS buffer, 5-20 vol% glycerol, 1-5 vol‰ protease inhibitor, 2-8 vol‰ lysozyme, 0.5-1 mM β-mercaptoethanol, and 50-100 U / 100 mL totipotent nuclease.

4. The purification method according to any one of claims 1 to 3, wherein, In step S02, the gradient washing includes: performing gradient washing on the total membrane proteins using a membrane protein washing solution with progressively increasing detergent concentration until no protein is detected in the supernatant, and collecting the precipitate after washing; Preferably, in the gradient washing, each gradient is incubated at 2-10°C for 10-60 min by rotation and then centrifuged; when no protein is detected in the supernatant, the precipitate is washed with a membrane protein washing solution without detergent, and then centrifuged. Preferably, the detergent is urea and / or sodium cholate; more preferably, the membrane protein washing solution comprises: 40-60 mM Tris-HCl, 1-5 mM ethylenediaminetetraacetic acid, 0.5-2 vol% Tween 100, 0.2-0.8 M NaCl, 2-8 M urea and / or 1-5 g / 100 mL sodium cholate; more preferably, the difference in urea concentration between two adjacent washes is 1-3 M; more preferably, the difference in sodium cholate concentration between two adjacent washes is 1-3 g / 100 mL.

5. The purification method according to any one of claims 1 to 4, wherein, In step S02, the denaturation and dissolution includes: contacting the precipitate after gradient washing with the protein denaturation solution to dissolve the target protein, separating and removing insoluble matter, and collecting the solution containing the denatured target protein; preferably, the dissolution of the target protein includes: rotating and incubating at 2-10°C for 1-3 hours. Preferably, in step S02, the dialysis refolding includes: placing a solution containing the denatured target protein in a dialysis bag, and performing dialysis in the protein dialysis refolding solution to refold the target protein; more preferably, the dialysis is performed at 2-10°C for 14 hours or more, preferably 14-20 hours. Preferably, in step S02, the ultrafiltration concentration includes: concentrating the dialyzed and refolded protein solution using an ultrafiltration tube; more preferably, the concentrated protein concentration is 5-10 mg / mL; Preferably, in step S02, the affinity chromatography includes: loading the concentrated protein solution onto an affinity chromatography column, sequentially performing column equilibration, loading, eluting contaminating proteins and eluting the target protein, and collecting the chromatographic eluent of the target protein; more preferably, the elution of contaminating proteins is performed using a buffer containing 20-80 mM imidazole; more preferably, the elution of the target protein is performed using a buffer containing 150-500 mM imidazole. Preferably, the C-terminus of the recombinant canine CYP450 protein is fused with a 6×His tag, and the affinity chromatography is nickel affinity chromatography.

6. A recombinant canine CYP450 protein, obtained by the purification method according to any one of claims 1 to 5.

7. A recombinant canine CYP450 protein formulation comprising the recombinant canine CYP450 protein of claim 6 or the recombinant canine CYP450 protein obtained by the purification method according to any one of claims 1 to 5.

8. A method for preparing a recombinant canine CYP450 protein formulation, wherein, The preparation method includes: ultrafiltration concentration, dialysis desalting, and cryopreservation of the chromatographic eluent of the target protein obtained by the purification method according to any one of claims 1 to 5.

9. The preparation method according to claim 8, wherein, The preparation method includes the following steps: ultrafiltration and concentration of the chromatographic eluent of the target protein to a protein concentration of 1-5 mg / mL, then placing it in a dialysis bag, dialyzing it in the dialysis solution to remove salt, then adding glycerol with a final concentration of 10-30% by volume as a cryoprotectant, and storing it at -70 to -90°C after quick freezing in liquid nitrogen. Preferably, the dialysis desalination is performed at 2-10°C for 14 hours or more, and more preferably for 14-20 hours.

10. Use of the recombinant canine CYP450 protein of claim 6 or the recombinant canine CYP450 protein formulation of claim 7 in the preparation of formulations for drug metabolism studies.

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