Uraurate oxidase mutant with high activity and high humanization rate

Abstract

The invention belongs to the technical field of biology, and relates to a urate oxidase mutant with high activity and high humanization rate. The urate oxidase mutant with high activity and high humanization rate is a human urate oxidase mutant obtained by mutating inactive human urate oxidase with an amino acid sequence of SEQ ID NO. 1, has 83rd, 119th, 121st, 151st, 222nd, 232, 233rd, 240th and 252nd amino acid substitutions, and also has 208th and 219th amino acid substitutions, and the human urate oxidase mutant is a human urate oxidase mutant obtained by mutating inactive human urate oxidase with an amino acid sequence of SEQ ID NO. And the compound further has 112th amino acid substitution. The urate oxidase mutant with high activity and high humanization rate keeps high homology with a theoretical amino acid sequence of human urate oxidase, keeps high activity at the same time, is more suitable for clinically treating hyperuricemia and gout for long-term use, and can be used for preparing drugs for treating hyperuricemia and gout.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a modified uricase, in particular to a uricase mutant with high activity and high humanization rate. Background Art

[0002] The main clinical treatment drugs for hyperuricemia and gout are allopurinol, xanthine oxidase inhibitors, benzbromarone, probenecid, and colchicine. These drugs generally have the risk of multi-organ toxicity, and their application is severely limited. Urate oxidase, as a key enzyme in the terminal link of purine metabolism, is responsible for catalyzing the conversion of uric acid into more soluble allantoin, showing a more ideal uric acid-lowering effect. Currently, the recombinant urate oxidase approved for the treatment of hyperuricemia in clinical practice is mainly derived from heterologous species, such as Aspergillus flavus and pig-baboon chimeras, which often cause severe allergic reactions in patients due to heterologousness. Although the half-life and stability of uricase modified with polyethylene glycol (PEG) have been improved, patients may still develop anti-drug antibodies (ADAs) and infusion reactions, which limits its wide clinical application. Therefore, it is mainly used to treat acute, severe, life-threatening and refractory hyperuricemia. The existence of immunogenicity has become the main limitation of uricase application. The development of uricase with low immunogenicity has become a hot area in the current research of recombinant uricase modification.

[0003] The human urate oxidase gene has become a pseudogene due to complex missense mutations and accumulation during evolution. Although short peptides of urate oxidase are found in human sweat glands, there is no urate oxidase activity. Jiang et al. reported that low-immunogenicity therapeutic urate oxidase was obtained by resurrecting the human urate oxidase pseudogene (Reference 1: Jiang N, Xuc, Zhang L, et al. “Resurrected” human-source urate oxidase with high uricolytic activity and stability [J]. Enzyme and Microbial Technology, 2021, 149: 109852.). The document shows that by performing multiple sequence alignment analysis on the inactive human uricase gene and mutating 15 highly conserved sites, a "resurrected" uricase rHU15 with a theoretical sequence identity of 95.06% with human uricase was obtained, but the enzyme activity was only 2.30U / mg. After further expanding the mutation of conservative sites, a mutant rHU19 with 19 sites was obtained (the theoretical sequence identity with human uricase was reduced to 93.75%), and the enzyme activity reached 8.29U / mg. The document did not test the immunogenicity of the mutant. Another document reported that even baboon uricase with a theoretical sequence identity of 93.75% with human uricase may cause significant immunogenicity (Document 2: Xiong Runsong. Study on cloning and molecular modification of baboon uricase gene [D]. Beijing University of Chemical Technology, 2012.). Compared with baboon uricase, rHU19 has lower sequence identity, so it can be inferred that rHU19 is more similar to heterologous uricase rather than human uricase, and still has significant immunogenicity. Since the human uricase pseudogene is revived by back mutation of all highly conserved sites, there is an essential non-humanization, which increases the heterologous nature of the revived enzyme and thus increases immunogenicity.

[0004] Human urate oxidase is a protein that has never existed in the body as a complete protein molecule during human development. There are two problems with its resurrection strategy: first, the activity of human urate oxidase is restored by mutation through conservation analysis of the amino acid sequences of isozymes from different species, which actually dehumanizes the protein amino acid sequence. Repair of conservative sites restores enzyme activity while enhancing immunogenicity. Second, when the humanization rate of the amino acid sequence reaches a threshold (for example, 95%), continuing to increase the humanization rate will lead to a significant decrease in enzyme activity or even loss of activity.

[0005] Therefore, the current strategy of functional resurrection of the human uricase pseudogene through homology alignment and conservative amino acid mutations faces challenges and difficulties. It is necessary to find new strategies to obtain human uricase resurrection mutants with high activity and high amino acid sequence humanization rate. Summary of the invention

[0006] The primary purpose of the present invention is to provide a highly active and highly humanized uricase mutant, which has a high homology with the theoretical amino acid sequence of human uricase and maintains a high activity.

[0007] The highly active and highly humanized uricase mutant of the present invention is a human uricase mutant obtained by mutation of an inactive human uricase with an amino acid sequence of SEQID NO. 1, and has amino acid substitutions at positions 83, 119, 121, 151, 222, 232, 233, 240 and 252.

[0008] According to the further characteristics of the highly active and highly humanized uricase mutant of the present invention, the amino acid substitution at position 83 is a substitution of glutamic acid (E) with glycine (G), the amino acid substitution at position 119 is a substitution of histidine (H) with arginine (R), the amino acid substitution at position 121 is a substitution of glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is a substitution of glutamine (Q) with proline (P), the amino acid substitution at position 222 is a substitution of serine (S) with phenylalanine (F), the amino acid substitution at position 232 is a substitution of leucine (L) with serine (S), the amino acid substitution at position 233 is a substitution of threonine (T) with proline (P), the amino acid substitution at position 240 is a substitution of cysteine ​​(C) with tyrosine (Y), and the amino acid substitution at position 252 is a substitution of alanine (A) with glutamic acid (E); the amino acid sequence of the uricase mutant is SEQ ID NO.2.

[0009] Optionally, the highly active and highly humanized uricase mutant of the present invention further has amino acid substitutions at positions 208 and 219.

[0010] According to further features of the highly active and highly humanized uricase mutant of the present invention, the amino acid substitution at position 208 is a substitution of lysine (K) with glutamic acid (E), and the amino acid substitution at position 219 is a substitution of methionine (M) with leucine (L); the amino acid sequence of the uricase mutant is SEQ ID NO.3.

[0011] Furthermore, the highly active and highly humanized uricase mutant of the present invention also has a substitution of the 112th amino acid.

[0012] According to a further feature of the highly active and highly humanized uricase mutant of the present invention, the amino acid substitution at position 112 is substitution of methionine (M) with valine (V); the amino acid sequence of the uricase mutant is SEQ ID NO.4.

[0013] The second object of the present invention is to provide a DNA molecule encoding the highly active and highly humanized uricase mutant of the present invention. When the amino acid sequence of the human uricase mutant described in the present invention is SEQ ID NO. 2, the nucleotide sequence of the human uricase mutant is SEQ ID NO. 5.

[0014] When the amino acid sequence of the human uricase mutant described in the present invention is SEQ ID NO. 3, the nucleotide sequence of the human uricase mutant is SEQ ID NO. 6.

[0015] When the amino acid sequence of the human uricase mutant described in the present invention is SEQ ID NO. 4, the nucleotide sequence of the human uricase mutant is SEQ ID NO. 7.

[0016] The third object of the present invention is to provide a vector containing the DNA molecule of the present invention.

[0017] The fourth object of the present invention is to provide a host cell, which contains the DNA molecule of the present invention, or contains the vector of the present invention.

[0018] The above-mentioned vectors and host cells can be prepared by techniques well known in the art.

[0019] The fifth object of the present invention is to provide a method for producing a uricase mutant with high activity and high humanization rate.

[0020] The method for producing the highly active and highly humanized uricase mutant of the present invention comprises: culturing the host cell of the present invention under conditions suitable for the expression of the humanized uricase mutant, and isolating the highly active and highly humanized uricase mutant from the culture medium.

[0021] When the DNA molecule of the present invention is inserted into the vector or transferred into the host cell in the appropriate orientation and correct reading frame, the DNA molecule can be expressed in any eukaryotic or prokaryotic expression system. Many host-vector systems can be used to express protein coding sequences. Host-vector systems include, but are not limited to: bacteria transformed with bacteriophages, plasmids or cosmids; microorganisms containing yeast vectors, such as yeast; mammalian cell systems infected with viruses; insect cell systems infected with viruses; plant cell systems infected with bacteria. Preferred vectors of the present invention include viral vectors, plasmids, cosmids or oligonucleotides.

[0022] The preferred host of the present invention is a prokaryotic system such as Escherichia coli; the preferred protein expression method of the present invention is expression in Bacillus.

[0023] The present invention is a human urate oxidase pseudogene (called HkDJ The amino acid sequence of the inactive human uricase gene was subjected to site-directed mutagenesis. The GENBANK accession number of the human uricase pseudogene sequence is NR_003927.2. The terminators at positions 33 and 187 in the sequence were changed to R to obtain the amino acid sequence of the inactive human uricase (SEQ ID NO.1).

[0024] The inventors carried out a mutated modification on the basis of SEQ ID NO.1 and obtained highly active and highly humanized mutants of human uricase mut1 (SEQ ID NO.2), mut2 (SEQ ID NO.3) and mut3 (SEQ ID NO.4), which have 96.38%, 95.72% and 95.39% homologies with the theoretical amino acid sequence of human uricase, respectively, and enzyme specific activities of 8.19 U / mg, 7.32 U / mg and 7.15 U / mg, respectively, which have a higher humanization rate than the existing publicly reported uricase, and show higher enzyme activity while breaking through the 95% homology threshold reported in the literature, and are therefore more suitable for the development of human uricase for clinical treatment and drugs for the treatment of hyperuricemia and gout in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 SDS-PAGE protein electrophoresis diagram of highly active human urate oxidase mutant, 1: rhUOX wt ; 2: rHU15 mutant; 3: mut1 mutant; 4: mut2 mutant; 5: mut3 mutant; M is a marker.

[0026] Figure 2 The rhUOX of the present invention is shown wt , rHU15, mut1, mut2 and mut3's uric acid degradation activity. DETAILED DESCRIPTION

[0027] The terms used herein, unless otherwise specified, have the meanings commonly understood by those skilled in the art. The following provides definitions of some special terms used in the present invention.

[0028] “rhUOX wt " indicates an inactive human uricase in which the missense (terminator) codons at positions 33 and 187 of the human uricase pseudogene are restored to arginine (R). The gene is in italics" rhuox wt "Indicates. wt The amino acid sequence is SEQ ID NO.1.

[0029] “rHU15” stands for rhUOX wt For the human urate oxidase control whose activity was revived by amino acid mutation, see the literature 1 of JIANG et al., whose gene is in italics " rhu15 "express.

[0030] "mut1" indicates the modified mut1 mutant of the present invention, and its gene is in italics mut1 ” indicates that the amino acid sequence of the mut1 mutant is SEQ ID NO.2.

[0031] "mut2" indicates the modified mut2 mutant of the present invention, and its gene is in italics mut2 ” indicates that the amino acid sequence of the Mut2 mutant is SEQ ID NO.3.

[0032] "mut3" indicates the modified mut3 mutant of the present invention, and its gene is in italics mut3 ” indicates that the amino acid sequence of the Mut3 mutant is SEQ ID NO.4.

[0033] Embodiment 1: rhuox wt , rhu15 , mut1 , mut2 and mut3 Gene synthesis The present invention adopts the human uricase pseudogene sequence (GenBank registration number is NR_003927.2) and obtains the human uricase mutant by transformation. rhuox wt , rhu15 , mut1 , mut2 and mut3The mutant genes were synthesized by BGI. The genes were cloned between NcoI and XhoI of pET28a plasmid (Invitrogen) and named pET28a-huox wt , pET28a-rhu15, pET28a-mut1 and pET28a-mut3.

[0034] Embodiment 2: rhuox wt , rhu15 , mut1 , mut2 and mut3 Genes were transformed into E. coli Rosetta (DE3) and recombinant protein was induced to express The recipient bacteria in this experiment was Escherichia coli Rosetta (DE3), and pET28a-huox wt After transformation with pET28a-rhu15, pET28a-mut1 and pET28a-mut3 recombinant plasmids, a preliminary screening was performed using a kanamycin (Kan) resistance plate. Single clones on the Kan plate were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h, after which the plasmids were extracted for PCR verification and further screening of positive clone recombinants.

[0035] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and cultured overnight at 37°C and 200 rpm. The revived bacterial solution was inoculated into 500 mL of LB medium (50 mg / mL Kan) at a 1% inoculation rate, cultured at 37°C and 200 rpm until OD600 was 0.6, and 60 μM lactose was added to induce expression at 30°C and 200 rpm for 12 hours. After induction, the bacteria were collected and resuspended in carbonate buffer at pH 10.0 (mass-to-volume ratio of 1:20), and the bacteria were broken by ultrasonic disruptor, centrifuged at 6000 g for 10 minutes, and the supernatant was collected to obtain the recombinant protein.

[0036] rhUOX will be obtained from the positive clone recombinants wt , rHU15, mut1, mut2 and mut3 mutant recombinant proteins were used in the experiments of Example 3 and Example 4 below.

[0037] Example 3: rhUOX wt SDS-PAGE electrophoresis detection of recombinant proteins of rHU15, mut1, mut2 and mut3 mutants The SDS-PAGE electrophoresis detection of the present embodiment comprises the following steps: (1) Prepare 10 mL of 10% separation gel, mix well and use a micropipette to pour the gel into the glass plate until it stops 2 to 3 cm from the upper edge of the short glass plate. Then seal the gel surface with distilled water. Gently lift one end of the gel dispenser and then put it down to make the gel surface flat. After polymerizing for 40 min, discard the distilled water and absorb excess water with filter paper.

[0038] (2) Prepare 4 mL of 5% concentrated gel and pour it evenly on the separation gel. Insert a comb of corresponding specifications while avoiding the generation of bubbles. Allow the gel to solidify for 30 min.

[0039] (3) Install the electrophoresis tank and fill it with electrophoresis liquid. The volume should be larger than half of the tank volume. Move the prepared gel into the tank and carefully remove the comb.

[0040] (4) Spot samples one by one. The amount of sample spotted should not be too much. 15 μL per well is appropriate.

[0041] (5) At the beginning of electrophoresis, set the voltage to 90 V. When the indicator reaches the concentrated gel, change the voltage to 120 V and continue electrophoresis. Stop electrophoresis when the target band reaches the middle position (the target band corresponds to the corresponding band of the Maker and can be known in advance).

[0042] (6) Carefully peel off the gel and stain with Coomassie Brilliant Blue R-250 for 30 min. Then destain with destaining buffer until the background is light and the protein bands are clear.

[0043] (7) Gel imaging and observation of results. The SDS-PAGE protein electrophoresis results are as follows: Figure 1 shown.

[0044] Figure 1 This is an SDS-PAGE protein electrophoresis diagram. Lane 1 is the denatured rhUOX wt Protein samples, lane 2 is the mutant rHU15 target protein sample. Lane 3 is the mutant mut1 target protein sample. Lane 4 is the mutant mut2 target protein sample. Lane 5 is the mutant mut3 target protein sample. The target protein size is about 34 kDa. This result shows that the target gene is successfully expressed in E. coli Rosetta and can be used for subsequent experiments.

[0045] Example 4: rhUOX wt Characterization of the activity of recombinant proteins of rHU15, mut1, mut2 and mut3 mutants At 37°C, take 20 μL of purified protein of appropriate concentration in 600 μL of 0.1M uric acid solution (pH 8.0), react for 10 minutes, and then add an equal volume of pure methanol to terminate the reaction. The enzyme activity is calculated by measuring the reduced ultraviolet absorption value at 293 nm by ultraviolet spectrophotometry. The control group is inactivated urate oxidase. The experimental results are shown in Figure 2 shown.

[0046] Enzyme activity unit definition: 1 unit is the amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute.

[0047] E (U / mg) = (△OD 293 × V1 × D) / (12.3 × V2 × T × C) Wherein, V1 is the total reaction volume; D is the dilution factor; 12.3 is the molar extinction coefficient of uric acid; T is the reaction time; and C is the protein concentration.

[0048] The comparison of the urate oxidase mutants described in the present invention with those in References 1 and 2 in terms of the number of point mutations, positions, homology, enzyme specific activity, etc. is shown in Table 1 below: Table 1:

[0049] Document 1: JIANG N, XU C, ZHANG L, et al. “Resurrected” human-source urateoxidase with high uricolytic activity and stability [J]. Enzyme and MicrobialTechnology, 2021, 149: 109852.

[0050] Reference 2: Xiong Runsong. Research on cloning and molecular modification of baboon urate oxidase gene[D]. Beijing University of Chemical Technology, 2012.

[0051] As can be seen from the above table, the present invention discloses for the first time highly active and highly humanized mutants mut1 (SEQ ID NO.2), mut2 (SEQ ID NO.3) and mut3 (SEQ ID NO.4), which have 96.38%, 95.72% and 95.39% homology to the theoretical amino acid sequence of human urate oxidase, respectively, and the corresponding activities are 8.19U / mg, 7.32U / mg and 7.15U / mg, respectively, with high activity retained while reducing the number of mutation sites. These urate oxidase mutants are higher in humanization rate and activity than rHU15 (Reference 1), and show higher enzyme activity while breaking through the 95% homology threshold reported in the literature, so they are more suitable for long-term use in the clinical treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

Claims

1. A highly active and highly humanized urate oxidase mutant, characterized in that: The mutant is a human uricase mutant obtained by mutation of an inactive human uricase with an amino acid sequence of SEQ ID NO. 1, and has amino acid substitutions at positions 83, 119, 121, 151, 222, 232, 233, 240 and 252.

2. The highly active and highly humanized uricase mutant according to claim 1, characterized in that: The amino acid substitution at position 83 is to replace glutamic acid (E) with glycine (G), the amino acid substitution at position 119 is to replace histidine (H) with arginine (R), the amino acid substitution at position 121 is to replace glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is to replace glutamine (Q) with proline (P), the amino acid substitution at position 222 is to replace serine (S) with phenylalanine (F), the amino acid substitution at position 232 is to replace leucine (L) with serine (S), the amino acid substitution at position 233 is to replace threonine (T) with proline (P), the amino acid substitution at position 240 is to replace cysteine ​​(C) with tyrosine (Y), and the amino acid substitution at position 252 is to replace alanine (A) with glutamic acid (E); the amino acid sequence of the uricase mutant is SEQ ID NO.

2.

3. The highly active and highly humanized uricase mutant according to claim 1, characterized in that: There are also amino acid substitutions at positions 208 and 219.

4. The highly active and highly humanized uricase mutant according to claim 3, characterized in that: The amino acid substitution at position 208 is to replace lysine (K) with glutamic acid (E), and the amino acid substitution at position 219 is to replace methionine (M) with leucine (L); the amino acid sequence of the uricase mutant is SEQ ID NO.

3.

5. The highly active and highly humanized uricase mutant according to claim 3, characterized in that: It also has an amino acid substitution at position 112.

6. The highly active and highly humanized uricase mutant according to claim 5, characterized in that: The amino acid substitution at position 112 is substitution of methionine (M) with valine (V); the amino acid sequence of the uricase mutant is SEQ ID NO.

4.

7. A DNA molecule, characterized in that: It encodes the highly active and highly humanized uricase mutant according to any one of claims 1 to 6.

8. The DNA molecule according to claim 7, characterized in that: The nucleotide sequences thereof are SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7 respectively.

9. A carrier, characterized in that: It contains the DNA molecule according to claim 7 or 8.

10. A host cell, characterized in that: It contains the DNA molecule according to claim 7 or 8, or contains the vector according to claim 9.

11. A method for producing a highly active and highly humanized urate oxidase mutant according to any one of claims 1 to 6, characterized in that: include: Cultivate the host cell according to claim 10 under conditions suitable for the expression of uricase, and separate the uricase mutant from the culture medium.

12. Use of the highly active and highly humanized urate oxidase mutant according to any one of claims 1 to 6 for preparing a drug for treating hyperuricemia and gout.

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