Human urate oxidase mutant and its preparation method and application

Abstract

The present invention provides a human urate oxidase mutant, a preparation method, and an application thereof. The amino acid sequence of the mutant is shown in SEQ ID NO.1 in the sequence listing. The mutant is prepared by (a) inclusion body denaturation pretreatment; (b) inclusion body denaturation; and (c) protein renaturation. The preparation process does not require a column chromatography step, saving production costs. The obtained mutant protein has high purity, has urate oxidase activity, can be used for oxidative degradation of uric acid, and has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to a human urate oxidase mutant and a preparation method and application thereof. Background Art

[0002] Hyperuricemia (HUA) is a globally prevalent metabolic disorder characterized by abnormally elevated serum uric acid (SUA) concentrations. Evolving dietary patterns have led to a surge in the intake of purine-rich meat, seafood, and animal offal. This metabolic conversion produces excessive uric acid, directly leading to an imbalance in serum uric acid homeostasis.

[0003] Hyperuricemia is closely associated with a variety of metabolic diseases, including gout, diabetes, hypertension, atherosclerosis, and chronic kidney disease. Lifestyle changes are the cornerstone of hyperuricemia management, including a low-purine diet, moderate exercise, weight control, and limited alcohol intake. However, lifestyle changes alone are often not enough to effectively control blood uric acid levels. Although existing uric acid-lowering drugs (such as allopurinol, febuxostat, benzbromarone, etc.) are effective to a certain extent, they have some limitations, such as adverse reactions, drug resistance, and poor efficacy in some patients.

[0004] Uricase, a key enzyme in uric acid metabolism, catalyzes the conversion of uric acid into the more water-soluble allantoin. However, during primate evolution, a series of mutations in the human urate oxidase gene resulted in its inactivity. While this evolutionary event conferred physiological advantages on humans, such as uric acid's antioxidant properties and blood pressure maintenance, it also increased susceptibility to metabolic diseases such as hyperuricemia and gout.

[0005] In recent years, through the combination of genetic engineering and protein modification technology, researchers have attempted to reconstruct active humanized urate oxidase mutants. However, traditional recombinant protein purification relies on chromatography column technology, which has complex equipment, expensive consumables and cumbersome operations, significantly increasing production costs. During column chromatography, the dynamic loading capacity limit of the filler requires frequent replacement or regeneration of the chromatography medium. In addition, multi-step operations such as column loading, equilibration, and elution require precise control of the flow rate and buffer system, which not only prolongs the production cycle, but also increases the labor cost of process validation and repetitive maintenance. In addition, the scale-up of the chromatography column requires re-optimization of parameters, resulting in a disconnect between R&D and production. The development of a new preparation process that does not require column chromatography can simplify the purification process, avoid filler loss and column efficiency attenuation problems, and systematically reduce production costs by reducing equipment investment, material consumption and process complexity. This has important economic value for improving the production efficiency of biological products and promoting industrial applications, and has become a key breakthrough in optimizing the biopharmaceutical technology system.

[0006] In summary, mutating the inactive human uricase natural protein into a human uricase oxidase mutant with uricase oxidation activity and establishing a low-cost preparation process that does not require a column chromatography step have important clinical significance. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a human urate oxidase mutant.

[0008] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned human urate oxidase mutant.

[0009] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned human urate oxidase mutant.

[0010] In order to solve the above technical problems, the technical solution of the present invention is:

[0011] A human urate oxidase mutant, the amino acid sequence of which is shown in the sequence listing SEQ ID NO.1.

[0012] The above-mentioned human uricase mutant protein is obtained by mutating 16 amino acid residues of human uricase, retains the human uricase framework to reduce immunogenicity, and has uricase activity.

[0013] Preferably, the nucleotide sequence of the human uricase mutant is shown in SEQ ID NO.2 in the sequence listing.

[0014] The preparation method of the human urate oxidase mutant is as follows:

[0015] (a) Pretreatment of inclusion bodies before denaturation

[0016] (1) Take the solution after lysis, centrifuge at 6000-8000 rpm, 0-4°C for 10-15 minutes, discard the supernatant to obtain the inclusion body precipitate, add inclusion body washing buffer, blow the precipitate until it is completely dissolved, and oscillate for 20-30 seconds every 10-15 minutes, and oscillate 2-3 times; the solution after lysis is a solution of bacterial engineered bacteria containing a sequence encoding a human urate oxidase mutant protein, and the inclusion body washing buffer contains 10-100 mM sodium phosphate buffer, 2 M urea, and a pH of 6.8-7.2;

[0017] (2) Centrifuge at 6000-8000 rpm and 0-4°C for 10-15 min, discard the supernatant, re-add inclusion body washing buffer, pipette the precipitate until completely dissolved, and oscillate for 20-30 seconds every 10-15 min, oscillating 2-3 times;

[0018] (3) Repeat step (2) 1-2 times, washing for a total of 1-2 hours;

[0019] (b) Inclusion body denaturation

[0020] After pretreatment, centrifuge at 6000-8000 rpm and 0-4°C for 10-15 min to obtain inclusion body precipitates, which are then resuspended completely with denaturing buffer, stirred overnight at 0-4°C and 60-100 rpm, and centrifuged at 10000-14000 rpm and 0-4°C for 15-25 min to separate soluble and insoluble proteins, and the supernatant is collected; the denaturing buffer contains 10-100 mM sodium phosphate buffer, 6-8 M urea, and a pH of 6.8-7.2;

[0021] (c) Protein renaturation

[0022] (1) The denatured protein was renatured at 2-4°C using a renaturation solution containing 10-100 mM sodium phosphate buffer, 0.1-0.5 mM L-arginine (L-Arg), 1-2 mM cysteine, 0.1-0.5 mM cystine, 3-7 mM EDTA, 0.3-0.7 mM guanidine hydrochloride, pH = 6.8-7.2, a protein concentration of 0.1-0.2 mg / mL, and a renaturation time of 10-15 hours;

[0023] (2) The renatured protein is subjected to buffer exchange by ultrafiltration to concentrate the protein concentration to 8-12 times the original concentration. The exchange buffer used contains 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine (L-Arg), and a pH of 6.8-7.2.

[0024] Preferably, the molecular weight of the recombinant human uricase mutant protein obtained by the above-mentioned method for preparing the human uricase mutant is about 35 kDa.

[0025] Application of the above-mentioned human uricase mutant in the oxidative degradation of uric acid.

[0026] Preferably, the method for oxidative degradation of uric acid using the human uricase mutant is as follows: uric acid solution is dissolved in potassium phosphate buffer, and human uricase mutant solution is added.

[0027] Beneficial effects:

[0028] By comparing the primary structures of ancestral uricase and human uricase, combined with crystal structure analysis, and based on the conserved folding and active site distribution of mammalian uricase, this paper designs a human uricase mutant with uricase activity, and proposes a method for preparing the uricase mutant protein based on inclusion body renaturation:

[0029] (1) Reconstruction of catalytic activity within the human enzyme framework through evolution-guided site-specific mutagenesis;

[0030] (2) Establish an efficient prokaryotic expression-renaturation system to solve the bottleneck of large-scale production of recombinant enzymes.

[0031] The human uricase mutant has uricase activity and the preparation method does not require a column chromatography step, resulting in high protein purity and saving production costs. Specifically:

[0032] (1) Based on evolutionary structural biology analysis, key functional domains were identified through multiple sequence alignment and X-ray crystallography. On this basis, a rational design strategy was adopted: the human uricase framework was retained to reduce immunogenicity, while key residues of the ancestral enzyme were introduced to restore catalytic efficiency. The resulting human uricase mutant possessed uricase activity.

[0033] (2) The preparation method is simple. By pre-treating the inclusion bodies, optimizing the protein concentration, and optimizing the pH and formulation of the refolding buffer, the mutant protein can be prepared without column chromatography. The purity is not less than 90%, the preparation cost is low, and it is suitable for scale-up production.

[0034] (3) The buffer solution of the prepared human urate oxidase mutant contains only sodium phosphate buffer and L-arginine, and does not contain urea, guanidine hydrochloride, etc. It has simple ingredients, low cost, and high safety for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A diagram showing the sequence alignment results of human uricase, ancestral uricase, and the human uricase mutant of the present invention;

[0036] Figure 2 This is the three-dimensional structure diagram of the human urate oxidase mutant;

[0037] Figure 3 This is the gel electrophoresis diagram of the massive expression of human urate oxidase mutant protein;

[0038] Figure 4 This is the gel electrophoresis image of human urate oxidase mutant protein after inclusion body denaturation pre-treatment;

[0039] Figure 5 This is the gel electrophoresis diagram of the denatured human urate oxidase mutant protein;

[0040] Figure 6 This is the gel electrophoresis diagram of the denatured protein of human urate oxidase mutant after renaturation;

[0041] Figure 7 This figure shows the results of identifying the activity of the denatured protein of human urate oxidase mutants. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0043] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0044] Example 1

[0045] Design of human urate oxidase mutants. The specific design ideas are as follows:

[0046] (1) Multiple sequence alignment: The amino acid sequences of dog, human, and ancestral urate oxidase were aligned using Clustal Omega, and the alignment results were analyzed using Jalview to identify conserved regions and functional domains;

[0047] (2) Rational design strategy: select residues near the substrate binding pocket as hotspot residues, design mutation sites based on multiple sequence alignment results, and retain the humanized framework;

[0048] (3) Immunogenicity prediction: NetMHCIIpan is used to predict potential T cell epitopes after mutation, and highly immunogenic sites are preferentially removed.

[0049] Finally, the optimal mutant amino acid sequence of the human uricase mutant was obtained as shown in the sequence listing SEQ ID NO.1. The alignment results of this sequence with the primary structure sequence of human uricase and ancestral uricase are shown in Figure 1 The mutation site is marked with a box; the three-dimensional structure of the human urate oxidase mutant is shown in Figure 2 The protein is a homotetrameric structure, and the main mutation site in one chain is marked with a sphere.

[0050] Example 2

[0051] The human urate oxidase mutant was prepared as follows:

[0052] (1) Pretreatment of inclusion body denaturation: Take the solution of genetically engineered bacteria (host cell is Escherichia coli BL21 (DE3)) containing the human urate oxidase mutant (amino acid sequence is shown in the sequence list SEQ ID NO.1, nucleotide sequence is shown in the sequence list SEQ ID NO.2) after lysis; centrifuge the engineered bacteria at 8000 rpm, 0-4℃ for 10 min, discard the supernatant to obtain the inclusion body precipitate, the engineered bacteria express a large amount of gel electrophoresis results as shown Figure 3As shown, it shows that the target protein was successfully expressed after fermentation of the genetically engineered bacteria, and was mainly present in the insoluble lysed precipitate sample, indicating that the protein existed in the form of inclusion bodies; an appropriate volume (about 1 / 10 of the fermentation broth volume) of inclusion body washing buffer (10mM sodium phosphate buffer, 2M urea, pH 6.8-7.2) was added to the inclusion body precipitate obtained by the above centrifugation, and the precipitate was pipetted until completely dissolved. Votex (high-frequency oscillation) was repeated for 20-30s every 15 minutes, and the operation was repeated for 0.5 hours (Votex 1-2 times); centrifuged at 8000 rpm, 0-4℃ for 10 minutes, the supernatant was discarded, and an equal volume of inclusion body washing buffer (1 / 10 of the fermentation broth volume) was added again. The precipitate was pipetted until completely dissolved. Votex (high-frequency oscillation) was repeated for 20-30s every 15 minutes, and the operation was repeated for 0.5 hours (Votex 1-2 times). The total washing time was 1.5 hours. The gel electrophoresis results of the human urate oxidase mutant inclusion body protein denaturation pre-treatment are shown as follows. Figure 4 As shown, it shows that most of the irrelevant proteins in the inclusion bodies have been removed through inclusion body denaturation pre-treatment.

[0053] (2) Denaturation of inclusion bodies: After pretreatment, centrifuge at 8000 rpm and 0-4°C for 10 min to obtain inclusion body precipitates, resuspend completely with denaturation buffer (10 mM sodium phosphate buffer, 8 M urea, pH 6.8-7.2), stir overnight at 4°C at low speed (60-100 rpm), centrifuge at 10000 rpm for 20 min to separate soluble and insoluble proteins, and take the supernatant, in which the target protein should be mainly present in the supernatant. Gel electrophoresis of human urate oxidase mutant protein after denaturation is as follows: Figure 5 As shown, the human urate oxidase mutant protein that had undergone inclusion body pre-treatment was successfully denatured under high concentration denaturant (8 M urea) conditions, and the denatured protein existed in a soluble form in the centrifugation supernatant.

[0054] (3) Protein renaturation: The denatured human urate oxidase mutant protein was renatured at 2-4°C (10 mM sodium phosphate buffer, 0.1 M L-arginine (L-Arg), 1 mM cysteine, 0.1 mM cystine, 5 mM EDTA, 0.5 mM guanidine hydrochloride, pH 7.0), with a protein concentration of 0.1-0.2 mg / mL and a renaturation time of 12 hours; the renatured protein was buffer-exchanged by ultrafiltration (buffer after replacement: 10 mM sodium phosphate buffer, 0.1 L-arginine (L-Arg), pH 7.0). Gel electrophoresis of the human urate oxidase mutant protein after renaturation is shown in Figure 2. Figure 6 As shown, a protein band with a molecular weight of approximately 35 kDa appeared in the supernatant after centrifugation, indicating that soluble protein can be obtained after the denatured protein of the human urate oxidase mutant is renatured according to the method of the present invention.

[0055] Example 3

[0056] Urate oxidase activity assay

[0057] To the system, 2 mL of a 10 mg / dL uric acid solution (dissolved in potassium phosphate buffer, pH 6.0) and varying volumes (0 μL, 10 μL, 20 μL, 40 μL, and 80 μL) of a renatured human urate oxidase mutant solution (concentration approximately 10 μg / mL) were added. The reaction was incubated at 37°C for 15 minutes. A blank control (no uric acid) was also set up, and the absorbance of the system was measured at 290 nm using a microplate reader. A graph was plotted with the absorbance of the system as the y-axis and the mass of the renatured human urate oxidase mutant protein as the abscissa.

[0058] The results are as follows Figure 7 As shown, after adding 0.1 μg of the renatured human uricase mutant protein prepared in Example 1, the absorbance at 290 nm dropped sharply, indicating that the human uricase mutant prepared by the scheme of the present invention has uricase activity, and the absorbance of uric acid at 290 nm dropped sharply, indicating that a very small amount of the renatured human uricase mutant protein solution can oxidize and degrade uric acid.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. The improvements and modifications made by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. A human urate oxidase mutant, characterized in that: Its amino acid sequence is shown in the sequence listing SEQ ID NO.

1.

2. The method for preparing the human urate oxidase mutant according to claim 1, characterized in that: The specific steps are as follows: (a) Pretreatment of inclusion bodies before denaturation (1) Take the solution after lysis, centrifuge at 6000-8000 rpm, 0-4°C for 10-15 minutes, discard the supernatant to obtain the inclusion body precipitate, add inclusion body washing buffer, blow the precipitate until it is completely dissolved, and oscillate for 20-30 seconds every 10-15 minutes, and oscillate 2-3 times; the solution after lysis is a solution of bacterial engineered bacteria containing a sequence encoding a human urate oxidase mutant protein, and the inclusion body washing buffer contains 10-100 mM sodium phosphate buffer, 2 M urea, and a pH of 6.8-7.2; (2) Centrifuge at 6000-8000 rpm and 0-4°C for 10-15 min, discard the supernatant, re-add inclusion body washing buffer, pipette the precipitate until completely dissolved, and oscillate for 20-30 seconds every 10-15 min, oscillating 2-3 times; (3) Repeat step (2) 1-2 times, washing for a total of 1-2 hours; (b) Inclusion body denaturation After pretreatment, centrifuge at 6000-8000 rpm and 0-4°C for 10-15 min to obtain inclusion body precipitates, which are then resuspended completely with denaturing buffer, stirred overnight at 0-4°C and 60-100 rpm, and centrifuged at 10000-14000 rpm and 0-4°C for 15-25 min to separate soluble and insoluble proteins, and the supernatant is collected; the denaturing buffer contains 10-100 mM sodium phosphate buffer, 6-8 M urea, and a pH of 6.8-7.2; (c) Protein renaturation (1) The denatured protein was renatured at 2-4°C using a renaturation solution containing 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine, 1-2 mM cysteine, 0.1-0.5 mM cystine, 3-7 mM EDTA, 0.3-0.7 mM guanidine hydrochloride, pH = 6.8-7.2, a protein concentration of 0.1-0.2 mg / mL, and a renaturation time of 10-15 hours; (2) The renatured protein is subjected to buffer exchange by ultrafiltration to concentrate the protein concentration to 8-12 times the original concentration. The exchange buffer used contains 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine, and a pH of 6.8-7.

2.

3. Use of the human uricase mutant according to claim 1 in the oxidative degradation of uric acid, wherein the use is for non-disease diagnosis and treatment purposes.

4. The use according to claim 3, characterized in that: The method for oxidative degradation of uric acid is as follows: uric acid solution is dissolved in potassium phosphate buffer, and human urate oxidase mutant solution is added.

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