Highly active and highly humanized urate oxidase mutants

Abstract

The application belongs to the technical field of biology and relates to a high-activity and high-humanization rate uric acid oxidase mutant. The high-activity and high-humanization rate uric acid oxidase mutant is a human uric acid oxidase mutant obtained by mutation of an inactivated human uric acid oxidase with an amino acid sequence of SEQ ID NO. 1, which has amino acid substitutions at positions 83, 119, 121, 151, 222, 232, 233, 240 and 252, also has amino acid substitutions at positions 208 and 219, and further has an amino acid substitution at position 112. The high-activity and high-humanization rate uric acid oxidase mutant has high homology with a theoretical amino acid sequence of human uric acid oxidase, has high activity, is more suitable for long-term use in the treatment of hyperuricemia and gout in the clinic, and can be used for preparing a drug for treating hyperuricemia and gout.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to an engineered uricase, in particular to a uricase mutant with high activity and high humanization rate. BACKGROUND

[0002] The main clinical treatment drugs for hyperuricemia and gout are allopurinol, xanthine oxidase inhibitors, benzbromarone, probenecid, colchicine, etc. These drugs generally have the risk of multi-organ toxicity, and their application is severely limited. Uricase is a key enzyme in the terminal stage of purine metabolism, which is responsible for catalyzing the conversion of uric acid into allantoin with higher solubility, showing a more ideal effect of lowering uric acid. Currently, the recombinant uricase approved for the treatment of hyperuricemia in the clinic mainly comes from heterologous species such as Aspergillus flavus and pig-baboon chimeras, which often causes serious allergic reactions in patients due to heterogeneity. Although the half-life and stability of the PEG-modified uricase are improved, patients may still produce anti-drug antibodies (ADAs) and infusion reactions, thereby limiting its widespread application in the clinic, and it is mainly used for the treatment of acute, severe, life-threatening refractory hyperuricemia. The presence of immunogenicity has become the main limitation of the application of uricase. The development of uricase with low immunogenicity has become a hot research field of the current 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 a low immunogenicity therapeutic urate oxidase was obtained by reviving the human urate oxidase pseudogene (Document 1: JIANG N, XU C, 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 analyzing the multiple sequence alignment of the inactive human urate oxidase gene, mutations were made at 15 highly conserved sites, obtaining a “revived” urate oxidase rHU15 with a sequence consistency of 95.06% with the theoretical sequence of human urate oxidase amino acid. However, the specific enzyme activity was only 2.30 U / mg. Further mutation of the expanded conserved site obtained a mutant rHU19 with 19 sites (the sequence consistency with the theoretical sequence of human urate oxidase amino acid decreased to 93.75%), and the specific enzyme activity reached 8.29 U / mg. The document did not test the immunogenicity of the mutant. Another document reports that even the baboon urate oxidase with a sequence consistency of up to 93.75% with the theoretical sequence of human urate oxidase amino acid can cause significant immunogenicity (Document 2: XIONG R, Cloning and molecular modification of baboon urate oxidase gene [D]. Beijing University of Chemical Technology, 2012.). Compared with baboon urate oxidase, rHU19 has lower sequence consistency, so it can be inferred that rHU19 is more similar to heterologous urate oxidase than to human origin, and still has significant immunogenicity. Since the human urate oxidase pseudogene is revived by back-mutation of all highly conserved sites, it is essentially non-human, increasing the heterogeneity of the revived enzyme and thus increasing the immunogenicity.

[0004] As a protein that has never existed in the form of a complete protein molecule in the human body during development, there are two problems with the revival strategy of human urate oxidase: first, by analyzing the conservation of amino acid sequences of isozymes from different species to restore the activity of human urate oxidase, the protein amino acid sequence is actually non-humanized, and the restoration of conserved sites enhances the immunogenicity while restoring enzyme activity; second, when the humanization rate of amino acid sequence reaches a threshold (e.g., 95%), further increasing the humanization rate actually leads to a significant decrease in enzyme activity, and even loss of activity.

[0005] Therefore, the current strategy of functional recovery of human urate oxidase pseudogene by homologous alignment and mutation of conserved amino acids is facing challenges and difficulties, and it is necessary to find new strategies to obtain high-activity and high-humanization rate of human urate oxidase mutant. SUMMARY

[0006] The primary object of the present application is to provide a high-activity and high-humanization rate of urate oxidase mutant which maintains high homology with the theoretical amino acid sequence of human urate oxidase while maintaining high activity.

[0007] The high-activity and high-humanization rate of urate oxidase mutant according to the present application is a human urate oxidase mutant obtained by mutation of the inactive human urate oxidase with the amino acid sequence of SEQ ID NO. 1, which has amino acid substitutions at positions 83, 119, 121, 151, 222, 232, 233, 240 and 252.

[0008] According to a further feature of the high-activity and high-humanization rate of urate oxidase mutant according to the present application, the amino acid substitution at position 83 is substitution of glutamic acid (E) with glycine (G), the amino acid substitution at position 119 is substitution of histidine (H) with arginine (R), the amino acid substitution at position 121 is substitution of glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is substitution of glutamine (Q) with proline (P), the amino acid substitution at position 222 is substitution of serine (S) with phenylalanine (F), the amino acid substitution at position 232 is substitution of leucine (L) with serine (S), the amino acid substitution at position 233 is substitution of threonine (T) with proline (P), the amino acid substitution at position 240 is substitution of cysteine (C) with tyrosine (Y), and the amino acid substitution at position 252 is substitution of alanine (A) with glutamic acid (E); the amino acid sequence of the urate oxidase mutant is SEQ ID NO. 2.

[0009] Alternatively, the high-activity and high-humanization rate of urate oxidase mutant according to the present application further has amino acid substitutions at positions 208 and 219.

[0010] According to a further feature of the high-activity and high-humanization rate of urate oxidase mutant according to the present application, the amino acid substitution at position 208 is substitution of lysine (K) with glutamic acid (E), and the amino acid substitution at position 219 is substitution of methionine (M) with leucine (L); the amino acid sequence of the urate oxidase mutant is SEQ ID NO. 3.

[0011] Further, the high-activity and high-humanization rate of urate oxidase mutant according to the present application further has an amino acid substitution at position 112.

[0012] According to a further feature of the high-activity and high-humanization rate urate oxidase mutant according to the present application, the amino acid substitution at position 112 is substitution of methionine (M) with valine (V); and the amino acid sequence of the urate oxidase mutant is SEQ ID NO. 4.

[0013] A second object of the present application is to provide a DNA molecule encoding the high-activity and high-humanization rate urate oxidase mutant according to the present application.

[0014] When the amino acid sequence of the human urate oxidase mutant according to the present application is SEQ ID NO. 2, the nucleotide sequence of the human urate oxidase mutant is SEQ ID NO. 5.

[0015] When the amino acid sequence of the human urate oxidase mutant according to the present application is SEQ ID NO. 3, the nucleotide sequence of the human urate oxidase mutant is SEQ ID NO. 6.

[0016] When the amino acid sequence of the human urate oxidase mutant according to the present application is SEQ ID NO. 4, the nucleotide sequence of the human urate oxidase mutant is SEQ ID NO. 7.

[0017] A third object of the present application is to provide a vector containing the DNA molecule according to the present application.

[0018] A fourth object of the present application is to provide a host cell containing the DNA molecule according to the present application, or containing the vector according to the present application.

[0019] The above-mentioned vector and host cell can be prepared by techniques well known in the art.

[0020] A fifth object of the present application is to provide a method for producing the high-activity and high-humanization rate urate oxidase mutant.

[0021] The method for producing the high-activity and high-humanization rate urate oxidase mutant according to the present application comprises culturing the host cell according to the present application under conditions suitable for expression of the human urate oxidase mutant, and isolating the high-activity and high-humanization rate urate oxidase mutant from the culture medium.

[0022] When the DNA molecule of the present application is inserted into the vector in a suitable orientation and correct reading frame, or is transferred into the host cell, the DNA molecule can be expressed in any eukaryotic or prokaryotic expression system. Many host-vector systems can be used to express the protein coding sequence. The host-vector systems include, but are not limited to, bacteria transformed with bacteriophage, plasmid or cosmid; microorganisms containing yeast vectors; mammalian cell systems infected with a virus; insect cell systems infected with a virus; plant cell systems infected with bacteria. The preferred vectors of the present application include viral vectors, plasmids, cosmids or oligonucleotides.

[0023] The preferred host of the present application is a prokaryotic system such as E. coli; the preferred protein expression method of the present application is bacillus expression.

[0024] The present application is a site-directed mutation of the amino acid sequence of the inactive human uric acid oxidase of the human uric acid oxidase pseudogene (referred to as hUOX The GENBANK accession number of the human uric acid oxidase pseudogene sequence is NR_003927.2. The stop codons at positions 33 and 187 in the sequence are changed to R, becoming the amino acid sequence of the inactive human uric acid oxidase (SEQ ID NO. 1).

[0025] Based on SEQ ID NO. 1, the inventors made a minimization modification to obtain the mutants mut1 (SEQ ID NO. 2), mut2 (SEQ ID NO. 3) and mut3 (SEQ ID NO. 4) of the human uric acid oxidase with high activity and high humanization, which have a homology of 96.38%, 95.72% and 95.39% with the theoretical amino acid sequence of human uric acid oxidase, respectively, and the specific enzyme activity is 8.19 U / mg, 7.32 U / mg and 7.15 U / mg, respectively. The humanization rate is higher than that of the existing publicly reported uric acid oxidase, and the homology threshold of 95% reported in the literature is broken, while showing high enzyme activity, so it is more suitable for developing human uric acid oxidase for clinical treatment and drugs for treating hyperuricemia and gout in the clinic. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 1 is a high-activity human uric acid oxidase mutant SDS-PAGE protein electrophoresis map, 1: rhUOX wt ; 2: rHU15 mutant; 3: mut1 mutant; 4: mut2 mutant; 5: mut3 mutant; M is Marker.

[0027] Figure 2 Figure 2 shows the degradation of uric acid activity of rhUOX wt , rHU15, mut1, mut2 and mut3 of the present application. Detailed Implementation

[0028] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art. Definitions of some specific terms used in this invention are provided below.

[0029] rhUOX wt "This indicates the inactive human uricase enzyme whose pseudogenes at positions 33 and 187 have been restored to arginine (R). The gene is in italics." rhuox wt "indicates". rhUOX wt The amino acid sequence is SEQ ID NO.1.

[0030] “rHU15” indicates rhUOX wt For the control of human uricase reactivated by amino acid mutation, see reference 1 by JIANG et al. (the gene is in italics). rhu15 "express.

[0031] "mut1" represents the modified mut1 mutant of this invention, whose gene is in italics. mut1 The '" indicates that the amino acid sequence of the mut1 mutant is SEQ ID NO.2.

[0032] "mut2" represents the mut2 mutant modified in this invention, whose gene is in italics. mut2 The amino acid sequence of the Mut2 mutant is SEQ ID NO.3.

[0033] "mut3" refers to the mut3 mutant modified in this invention, whose gene is in italics. mut3 The amino acid sequence of the Mut3 mutant is SEQ ID NO.4.

[0034] Example 1: rhuox wt , rhu15 , mut1 , mut2 and mut3 Gene synthesis

[0035] This invention uses a human uricase pseudogene sequence (GenBank accession number NR_003927.2) to modify and obtain a human uricase mutant. rhuox wt , rhu15 , mut1 , mut2 and mut3, the mutant genes were synthesized by Huada Gene Company. The genes were cloned between Ncol and Xhol of pET28a plasmid (Invitrogen Corporation), and were named as pET28a-huox wt , pET28a-rhu15, pET28a-mut1 and pET28a-mut3.

[0036] Example 2: rhuox wt 、 rhu15 、 mut1 、 mut2 and mut3 The genes were respectively transformed into E. coli Rosetta (DE3) and the induction expression of recombinant proteins

[0037] The receptor bacteria of this experiment were E. coli Rosetta (DE3), and after being transformed by pET28a-huox wt , pET28a-rhu15, pET28a-mut1 and pET28a-mut3 recombinant plasmids, kanamycin (Kan) resistant plates were used for preliminary screening, and then single colonies on the Kan plate were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h, and the plasmid was extracted for PCR verification, and the positive recombinant clones were further screened.

[0038] The positive recombinant clones were inoculated in 5 mL of LB medium (50 mg / mL Kan) and cultured at 37°C, 200 rpm for overnight. The recovered bacterial liquid was inoculated in 500 mL of LB medium (50 mg / mL Kan) at an inoculation amount of 1%, and cultured at 37°C, 200 rpm until the OD600 was 0.6. 60 μM lactose was added, and the expression was induced at 30°C, 200 rpm for 12 h. After induction, the bacterial cells were collected, the bacterial cells were resuspended with carbonate buffer at pH 10.0 (mass / volume ratio of 1:20), the bacterial cells were broken using an ultrasonic disrupter, centrifuged at 6000 g for 10 min, and the supernatant was collected, i.e. the recombinant protein was obtained.

[0039] The rhUOX wt , rHU15, mut1, mut2 and mut3 mutant recombinant proteins obtained from the positive recombinant clones were used in the experiments of Example 3 and Example 4.

[0040] Example 3: SDS-PAGE electrophoresis detection of rhUOX wt , rHU15, mut1, mut2 and mut3 mutant recombinant proteins

[0041] The SDS-PAGE electrophoresis detection of this example includes the following steps:

[0042] (1) Configuration 10 mL 10% separation gel, mix well with micro pipette to glass plate, until the short glass plate 2~3 cm from the top stop, then use distilled water to seal the gel surface, can gently lift the end of the gel device and then put down the gel surface flat, polymerization 40 min to discard distilled water, use filter paper to absorb excess water.

[0043] (2) Configuration 4 mL 5% concentrated gel, evenly filled in the separation gel, insert the corresponding size of the comb while avoiding the formation of bubbles, polymerization 30 min to be solidified.

[0044] (3) install electrophoresis tank, fill the tank with electrophoresis liquid, the volume should be greater than half of the volume of the electrophoresis tank, move the prepared gel into the electrophoresis tank, and carefully pull out the comb.

[0045] (4) in turn, the sample amount should not be too much, 15 μL per well is appropriate.

[0046] (5) electrophoresis starts with 90 V, the indicator to the concentrated gel part will change to 120 V, continue electrophoresis, when the target band reaches the middle position, stop electrophoresis (the target band corresponds to the corresponding band of Maker, which can be known in advance).

[0047] (6) carefully peel off the gel, coomassie brilliant blue R-250 staining for 30 min, and decoloring with decoloring solution until the background is light and the protein band is clear.

[0048] (7) gel imaging and observation of results. SDS-PAGE protein electrophoresis results are shown in Figure 1 .

[0049] Figure 1 is the SDS-PAGE protein electrophoresis map. Lane 1 is the denatured rhUOX wt protein sample, 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 size of the target protein is about 34 kDa. The results show that the target gene is successfully expressed in E. coli Rosetta, which can be used for subsequent experiments.

[0050] Example 4: Activity characterization of rhUOX wt , rHU15, mut1, mut2 and mut3 mutant recombinant proteins

[0051] At 37℃, 20 μL of purified protein of appropriate concentration was taken in 600 μL of 0.1 M uric acid solution (pH 8.0), and the reaction was terminated after 10 min by adding an equal volume of pure methanol. The enzyme activity was calculated by measuring the decrease in ultraviolet absorption at 293 nm by ultraviolet spectrophotometry. The control group was inactivated uric acid oxidase. The experimental results are shown in Table 1. Figure 2

[0052] Definition of enzyme activity unit: 1 unit is the amount of enzyme required to catalyze 1 μmol of uric acid oxidation per minute.

[0053] E (U / mg) = (△OD 293 × V1 × D) / (12.3 × V2 × T × C)

[0054] In the formula, V1 is the total reaction volume; D is the dilution multiple; 12.3 is the molar extinction coefficient of uric acid; T is the reaction time; and C is the protein concentration.

[0055] As for the number of point mutations, the position, homology, and enzyme specific activity, the uric acid oxidase mutant of the present application is compared with documents 1 and 2 in Table 1 below:

[0056] Table 1:

[0057]

[0058] Document 1: JIANG N, XU C, ZHANG L, et al. “Resurrected” human-source urate oxidase with high uricolytic activity and stability [J]. Enzyme and Microbial Technology, 2021, 149: 109852.

[0059] Document 2: Xiong Runsong. Chimpanzee uric acid oxidase gene cloning and molecular modification research [D]. Beijing University of Chemical Technology, 2012.

[0060] ​As can be seen from the above table, the present application first discloses the mutants mut1 (SEQ ID NO. 2), mut2 (SEQ ID NO. 3) and mut3 (SEQ ID NO. 4) with high activity and high humanization rate, the homology of which with the theoretical sequence of human uric acid oxidase amino acid is 96.38%, 95.72% and 95.39% respectively, and the corresponding activity is 8.19 U / mg, 7.32 U / mg and 7.15 U / mg respectively, and the activity is high while the mutation site is reduced. These uric acid oxidase mutants are higher than rHU15 (literature 1) in humanization rate and activity, break through the 95% homology threshold reported in the literature, and show higher enzyme activity, and are more suitable for long-term use in the treatment of hyperuricemia and gout in the clinic, and can be used for preparing drugs for treating hyperuricemia and gout.

Claims

1. A urate oxidase mutant having high activity and high humanization rate, characterized in that: The amino acid sequence of the uricase mutant is SEQ ID NO. 2 or SEQ ID NO.

3.

2. A DNA molecule characterized by: The DNA molecule of claim 2 or 3.

3. The DNA molecule of claim 2, wherein: The nucleotide sequence is SEQ ID NO. 5 or SEQ ID NO. 6, respectively.

4. A vector, characterized by: The DNA molecule of claim 2 or 3.

5. A host cell, characterized by: The DNA molecule of claim 2 or 3, or the vector of claim 4.

6. A method for producing a urate oxidase mutant having high activity and a high humanization rate according to claim 1, characterized by, Comprising: culturing the host cell of claim 5 under conditions suitable for expression of uricase, and isolating the uricase mutant from the culture medium.

7. Use of the high-activity and high-humanization rate uricase mutant of claim 1 for the preparation of a medicament for treating hyperuricemia and gout.

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