CN115197922B

CN115197922B - Uricase or its isoforms and methods of making same

Info

Publication number: CN115197922B
Application number: CN202110381755.0A
Authority: CN
Inventors: 徐敏; 王建辉; 王圣; 王春梅
Original assignee: Parker Biomedical Suzhou Co ltd
Current assignee: Parker Biomedical Suzhou Co ltd
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2024-05-14
Anticipated expiration: 2041-04-09
Also published as: CN115197922A

Abstract

The invention relates to the field of enzyme purification, in particular to uricase or an isoform thereof and a preparation method thereof. The invention adopts hydrophobic chromatography and anion exchange chromatography purification to purify uricase or an isoform thereof, the purity and specific activity of the uricase or the isoform thereof are obviously improved, and the uricase or the isoform thereof has high protein recovery rate and is suitable for industrial production.

Description

Uricase or its isoforms and methods of making same

Technical Field

The invention relates to the field of enzyme purification, in particular to uricase or an isoform thereof and a preparation method thereof.

Background

Uricase (EC 1.7.3.3), also known as urate oxidase, is a key enzyme for purine metabolism in organisms and can hydrolyze uric acid to water-soluble allantoin which is excreted from the body. Uricase is widely found in nature, including many mammals (swine, bovine, canine, murine, etc.), leguminous plants, and microorganisms, among others.

Primates, including humans, lack uricase in humans due to gene mutation, resulting in non-expression of uricase, which is the final product of purine metabolism in humans, and plasma uric acid levels in humans are approximately 10 times that of non-primates. Uric acid is a weak acid, under physiological conditions, most uric acid exists in the form of monosodium urate, and the solubility of monosodium urate is very low, so that hyperuricemia can be caused when the concentration of urate is high; high serum uric acid concentrations are a direct cause of gout, kidney stones and vascular diseases. In the long-time hyperuricemia state, urate is deposited in body tissues to cause damage and gout; the higher the blood uric acid level, the greater the likelihood of gout occurring in the next 5 years.

After McCarty and Hollander elucidated that the crystallization of monosodium uronate was the pathogenesis of gout from the 60 s of the last century, the search for uricase for treating gout has not ceased: extracting natural urate oxidase from Aspergillus flavus (Aspergillus flavus) in 1968 Laboureur P to treat tumor lysis syndrome; the FDA approved recombinant aspergillus flavus urate oxidase Rasburicase in 2002 was used to treat hyperuricemia caused by tumor lysis syndrome, but the search for attempted gout treatment was hampered due to its strong immunogenicity and short half-life; pegloticase was approved by the FDA in 2010 as a therapeutic agent for the treatment of refractory gout, which uses uricase of mammalian origin and pegylates it, in Pegloticase uricase being covalently bound to polyethylene glycol monomethyl ether [ mPEG ] (molecular weight 10 kDa), in order to solve the problems of high immunogenicity and short half-life of uricase Rasburicase at the time of treatment. Pegloticase, however, still has an impact on its clinical use due to immunogenicity problems, and it has been found in phase III clinical trials that 92% of patients develop anti-pegloticase antibodies, 42% of patients detect anti-PEG antibodies, and high anti-pegloticase antibodies not only affect uric acid lowering efficacy, but also cause allergic reactions (6.5%) in patients; in addition, pegloticase was administered by intravenous infusion and 26% of patients developed infusion reactions, including facial flushing, urticaria, hypotension, etc.

For uricase preparations, its immunogenicity stems from several factors: 1. uricase itself, such as uricase, is susceptible to aggregation, and the aggregated uricase is more immunogenic; if the biological activity of uricase is low, the low activity means that the dosage used for achieving the therapeutic effect is high, and the injection times are high, so that the antibody titer generated by the body is stimulated to be high; if uricase is exogenous, human body does not have uricase, all uricase is exogenous and can stimulate human antibody production; such as species-specific epitopes of uricase, the strength and number of T cell epitopes are also one of the important factors in determining uricase immunogenicity. 2. The composition of uricase preparation, and the route, dosage and frequency of administration. 3. Patient individual factors.

By combining the above factors, the new generation uricase preparation needs to have the characteristics of high activity, less dosage, less administration times, good solubility, difficult aggregation, few antigenic determinants and the like, so that safer and more effective treatment can be achieved. Another powerful means is to enhance the directional or targeted action of uricase at the tophus site, such as the local release of uricase at the tophus, which can accelerate the dissolution of uricase. The enhancement of the therapeutic effect can shorten the administration period, and reduce the administration dosage and the infusion frequency, thereby preventing and reducing the immune response. At present, uricase products are not marketed in China.

In the prior art, in order to obtain uricase and an allosteric thereof from various biological sources, engineering bacteria are generally utilized for expression and fermentation, and fermentation liquor is purified, and during purification, a plurality of composite means such as salting out, ultrafiltration, dialysis, affinity chromatography, ion exchange, hydrophobic chromatography, gel filtration and the like are mostly needed, so that the operation is complex, the protein recovery rate is low, the method is not suitable for industrial production, and the biological activity of uricase is even influenced by partial severe purification conditions. In view of the foregoing, there is a need in the art for a highly efficient and convenient method for purifying uricase that maximizes enzyme activity.

Disclosure of Invention

In view of this, the present invention provides uricase and methods of preparing the same. The purification method is optimized, so that the purity, recovery rate and specific activity of the uricase purified product are improved.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a method for purifying uricase or an isoform thereof, which comprises the steps of purifying a sample to be purified containing uricase or an isoform thereof by hydrophobic chromatography and anion exchange chromatography, respectively.

The hydrophobic chromatography and anion exchange chromatography are not strictly sequential, e.g., in one embodiment, the sample to be purified is subjected to hydrophobic chromatography followed by anion exchange chromatography; in yet another embodiment, the sample to be purified is subjected to anion exchange chromatography followed by hydrophobic chromatography. It will be appreciated by those skilled in the art that between the two above-described analyses, there may optionally be included operations of collection, concentration, reconstitution, dilution, etc.

In some embodiments of the present invention, samples purified via hydrophobic chromatography and anion exchange chromatography yield satisfactory purity and/or enzyme activity without further purification, including, but not limited to, salting out, ultrafiltration, dialysis, ion exchange, hydrophobic chromatography, gel chromatography (also known as molecular sieve or exclusion chromatography), or combinations thereof.

In some embodiments of the invention, the purified uricase or an isoform thereof may have a purity of greater than 85%, preferably greater than 90%, 91%, 92%, 93%, or 94%, more preferably greater than 95%, 96%, 97%, or 98%, even more preferably greater than 99%.

In some embodiments of the invention, prior to purification, further comprising a step of sample treatment comprising mixing the sample treatment solution with the sample to be purified and collecting crude uricase or an isoform thereof for use in the purification step; the sample processing solution includes a salt. Any salt consisting of a metal cation (or ammonium ion) and an anion may be used in the sample processing solution, and in some specific embodiments, the anion of the salt may be selected from one or more of PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-、SO₄ ^2-、CH₃COO^-、F^-、Cl^-、Br^-、NO₃ ^-、ClO₄ ^-、I^- and SCN ^-, as is commonly understood in the art; the cation of the salt may be selected from one or more of NH₄ ⁺、Rb⁺、K⁺、Na⁺、Cs⁺、Li⁺、Mg²⁺、Ca²⁺ and Ba ²⁺.

In particular, exemplary salts may be selected from one or more of Na₃PO₄、K₃PO₄、Na₂HPO₄、K₂HPO₄、NaH₂PO₄、KH₂PO₄、(NH₄)₂SO₄、Na₂SO₄、K₂SO₄、CH₃COONa、CH₃COOK、NaF、KF、CsF、NH₄Cl、NaCl、KCl、NaBr、KBr、NaI and KI; preferably (one or more of NH ₄)₂SO₄、Na₂SO₄、K₂SO₄、NH₄ Cl, naCl and KCl; more preferably (one or more of NH ₄)₂SO₄, naCl and KCl; even more preferably (NH ₄)₂SO₄).

The crude uricase or its isoforms may be collected by solid-liquid separation means conventional in the art, including but not limited to filtration, salting out, sedimentation, flocculation, centrifugation, etc., and the collected objects may be selected as appropriate according to the enriched site of the target protein, such as supernatant or precipitate, etc.; in a specific embodiment, the crude uricase or an isoform thereof is collected by centrifugation followed by collection of the supernatant. The temperature of the centrifugation may be 0 to 50 ℃, preferably 0 to 10 ℃, more preferably 0 to 4 ℃; the rotational speed of the centrifugation may be 1000-100000 rpm, and the time of the centrifugation may be 30 s-30 min.

In some embodiments of the invention, the salt concentration in the mixture after mixing the sample treatment solution with the sample to be purified is 0.05-3M, and may be 0.05M、0.1M、0.15M、0.2M、0.25M、0.3M、0.35M、0.4M、0.45M、0.5M、0.55M、0.6M、0.65M、0.7M、0.75M、0.8M、0.85M、0.9M、0.95M、1M、1.1M、1.15M、1.2M、1.25M、1.3M、1.4M、1.5M、1.6M、1.7M、1.8M、1.9M、2M、2.1M、2.2M、2.3M、2.4M、2.5M、2.6M、2.7M、2.8M、2.9M、3M;, preferably 0.2-2M; more preferably 0.5 to 1.5M; even more preferably 0.75-1.25M.

In some embodiments of the invention, the hydrophobic chromatography comprises the steps of first equilibration, loading, second equilibration, eluting impurities, and protein digestion; the mobile phase used in the first equilibration, second equilibration, elution impurities and protein digestion steps comprises salts.

In accordance with the general understanding in the art, any salt consisting of a metal cation (or ammonium ion) and an anion may be used in the mobile phase of the hydrophobic chromatography, in some specific embodiments, the anion of the salt may be selected from one or more of PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-、SO₄ ^2-、CH₃COO^-、F^-、Cl^-、Br^-、NO₃ ^-、ClO₄ ^-、I^- and SCN ^-; the cation of the salt may be selected from one or more of NH₄ ⁺、Rb⁺、K⁺、Na⁺、Cs⁺、Li⁺、Mg²⁺、Ca²⁺ and Ba ²⁺.

The mobile phase may also comprise a buffer solution of dissolved salts selected from one or more of borax borate buffer, phosphate buffer, tris buffer or citric acid buffer, preferably borax borate buffer;

The pH of the buffer solution is 7 to 12, and may be specifically 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, preferably 8 to 10, more preferably 8 to 9, even more preferably 8.5; there is no particular requirement for the concentration of the buffer solution during chromatography, for example, the concentration of the buffer solution may be 10 to 200mM, preferably 20 to 100mM, more preferably 30 to 70mM, and even more preferably 50mM.

In the hydrophobic chromatography process, the concentration of salt in the mobile phase is adjusted to adjust the hydrophobic action intensity and adsorption tightness of target proteins and other undesired substances and a chromatographic column so as to achieve the purpose of separation.

In some embodiments, the salt concentration in the mobile phase used in the second equilibration, elution impurities, and protein dissolution steps decreases sequentially. In particular, the salt concentration of the second equilibrium mobile phase may be in the range of 0.2 to 5M, preferably 0.5 to 3M, more preferably 1.5 to 2.5M, even more preferably 1.8M to 2.2M; the eluted impurity mobile phase may have a salt concentration of 0.1 to 4M, preferably 0.4 to 2.5M, more preferably 0.5 to 1.5M, even more preferably 0.8M to 1.2M; the salt concentration of the protein-eluting mobile phase is 0.01 to 3M, preferably 0.05 to 2M, more preferably 0.1 to 1M, even more preferably 0.2 to 0.8M. When the hydrophobic chromatography is used for purification, a person skilled in the art can select and combine the salt concentration of the mobile phase in each stage within the range, and the condition that the salt concentration is sequentially reduced is only required to be met.

In some embodiments, the flow rate of the hydrophobic chromatography is from 0.5 to 15ml/min, preferably from 1 to 10ml/min, more preferably from 2 to 8ml/min, even more preferably 5ml/min.

The hydrophobic chromatography of the invention has no special requirement on the used hydrophobic filler, and all fillers which can generate hydrophobic effect with hydrophobic groups in protein are in the considered range; in some embodiments of the invention, the filler of hydrophobic chromatography comprises one or more of OctylSepharose 4FastFlow, butyl Sepharose 4FastFlow, phenylSepharose 6Fast Flow or PhenylSepharose CL-4B; preferably, the filler for hydrophobic chromatography is Phenyl Sepharose 6FastFlow.

In some embodiments of the invention, the hydrophobic chromatography comprises the steps of:

And (3) filling: a PhenylSepharose 6FastFlow (high sub) chromatography packing was loaded into an empty chromatography column, column Volume (CV) 20ml purification step:

Balance: adding 50mM borax borate buffer solution pH 8.5 dissolved 2M (NH ₄)₂SO₄ solution, 5ml/min flowing through 3-5CV balance column

Loading: adding 100ml of treated sample, and loading at 5ml/min

Balance: adding 50mM borax borate buffer solution pH 8.5 dissolved 2M (NH ₄)₂SO₄ solution, flowing through the column at 5ml/min for 3-5CV to equilibrate the column again

Eluting: adding 50mM borax borate buffer solution pH 8.5 to dissolve 900mM (NH ₄)₂SO₄ solution, 5ml/min for eluting impurities

And (3) dissolving out: 500mM (NH ₄)₂SO₄ solution, 5 ml/min) of borax buffer solution at 50mM pH 8.5 was added to the solution, and the target protein was eluted, and the elution peak was collected.

In some embodiments of the present invention, prior to anion exchange chromatography purification, a step of sample treatment is further included, wherein the sample treatment is aimed at reducing the salt concentration in the sample to be purified, and any procedure that can achieve the above-mentioned aim can be used, including but not limited to: dilution, ultrafiltration, dialysis, chromatography, etc., and can be selected by the skilled in the art according to the size of the protein molecule and the actual need;

In a specific embodiment, the sample is processed by dilution means comprising: taking protein to be purified by anion exchange chromatography, and diluting the protein by 0 to 30 times by using a buffer solution; preferably diluted 5-20 times; more preferably 10-fold dilution;

the buffer solution comprises one or more of borax borate buffer solution, phosphate buffer solution, tris buffer solution and citric acid buffer solution.

The concentration of buffer used is 10 to 200mM, preferably 20 to 100mM, more preferably 30 to 70mM, even more preferably 50mM; the pH of the buffer solution is 7 to 12, preferably 8 to 10, more preferably 8.5;

in some embodiments of the invention, the anion exchange chromatography comprises the steps of first equilibration, loading, second equilibration, eluting impurities, and protein digestion; the mobile phase used in the eluting impurities and protein elution step contains salts.

In accordance with the general understanding in the art, any salt consisting of a metal cation (or ammonium ion) and an anion may be used in the mobile phase of the anion exchange chromatography, in some specific embodiments, the anion of the salt may be selected from one or more of PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-、SO₄ ^2-、CH₃COO^-、F^-、Cl^-、Br^-、NO₃ ^-、ClO₄ ^-、I^- and SCN ^-; the cation of the salt may be selected from one or more of NH₄ ⁺、Rb⁺、K⁺、Na⁺、Cs⁺、Li⁺、Mg²⁺、Ca²⁺ and Ba ²⁺.

In particular, exemplary salts may be selected from one or more of Na₃PO₄、K₃PO₄、Na₂HPO₄、K₂HPO₄、NaH₂PO₄、KH₂PO₄、(NH₄)₂SO₄、Na₂SO₄、K₂SO₄、CH₃COONa、CH₃COOK、NaF、KF、CsF、NH₄Cl、NaCl、KCl、NaBr、KBr、NaI and KI; preferably (one or more of NH ₄)₂SO₄、Na₂SO₄、K₂SO₄、NH₄ Cl, naCl and KCl; more preferably (one or more of NH ₄)₂SO₄, naCl and KCl; even more preferably NaCl).

The mobile phase also comprises a buffer solution for dissolving salt, and the buffer solution can be independently used as the mobile phase in the first balanced mobile phase and the second balanced mobile phase without salt, wherein the buffer solution is one or more selected from borax borate buffer solution, phosphate buffer solution, tris buffer solution or citric acid buffer solution, and preferably the borax borate buffer solution is used;

In the anion exchange chromatography process, the ion action intensity and the adsorption tightness between the target protein and other undesired substances and the chromatographic column are adjusted by adjusting the salt concentration in the mobile phase so as to achieve the purpose of separation.

In some embodiments, the salt concentration in the mobile phase used in the protein elution step is higher than in the impurity elution step during anion exchange chromatography. In particular, the salt concentration of the eluting impurity mobile phase may be in the range of 0.005 to 1M, preferably 0.01 to 0.5M, more preferably 0.05 to 0.3M, even more preferably 0.1 to 0.2M; the concentration of the protein-eluting mobile phase is 0.01 to 2M, preferably 0.05 to 1M, more preferably 0.1 to 0.5M, even more preferably 0.2 to 0.3M. When anion exchange chromatography is used for purification, a person skilled in the art can select and combine the salt concentration of the mobile phase in each stage within the above range, and only the salt concentration in the protein dissolution step is required to be higher than that in the impurity elution step.

In some embodiments, the anion exchange chromatography is performed at a flow rate of 0.2 to 10ml/min, preferably 0.5 to 8ml/min, more preferably 1 to 5ml/min, even more preferably 2ml/min.

The anion exchange chromatography of the invention has no special requirements on the used filler, and all fillers which can have ionic action with negatively charged groups in proteins are considered. In some embodiments of the invention, the packing employed in the anion exchange chromatography comprises one or more of Q Sepharose FF, DEAE Sepharose FF, capto Q, capto DEAE, capto sphere, or MacroCap Q; preferably, the filler used for the anion exchange chromatography is macroCap Q.

In some embodiments of the invention, the anion exchange chromatography comprises the steps of:

And (3) filling: packing a macroCap Q chromatography packing into an empty chromatography column, column Volume (CV) 5ml

And (3) purification:

Balance: adding 50mM borax borate buffer solution with pH of 8.5, and flowing through the column at 2ml/min for 3-5CV balancing column

Loading: adding the treated sample, and loading the sample by 2ml/min flowing through the column

Balance: 50mM borax borate buffer solution pH 8.5 was added and the column was equilibrated again by flowing through the column at 2ml/min at 3-5CV

Eluting: adding 150mM NaCl solution dissolved in 50mM borax borate buffer solution with pH of 8.5, and eluting impurities by 2ml/min flowing through the column

And (3) dissolving out: 50mM borax borate buffer solution pH 8.5 was added to the solution, and the solution was passed through the column at 2ml/min to dissolve the target protein, and the elution peak was collected.

Based on the above research, the invention also provides a preparation method of uricase or an isoform thereof, comprising the steps of:

Step 1, obtaining a sample to be tested containing uricase or an isoform thereof;

Step 2. Purification according to the purification method of the present invention gives uricase or an isoform thereof having a purity of 85% or more, preferably 90% or more, more preferably 95% or more.

In addition, the invention also provides uricase or an isoform thereof prepared according to the purification method or the preparation method.

The invention also provides a protein comprising the amino acid sequence of SEQ ID NO:1-8, or a sequence having an identity of 95%, 96%, 97%, 98% or 99% or more to the above sequence, the protein having a purity of 95% or more, preferably 96% or more, more preferably 97% or more, more preferably 98% or more, even more preferably 99% or more to the existing similar protein.

In some embodiments, the protein is prepared according to the purification methods described herein or the preparation methods described herein.

Based on the above research, the invention also provides a preparation method of the uricase or the conjugate of the isoform thereof, which comprises the step of conjugating the uricase or the isoform thereof with a polymer, wherein the uricase or the isoform thereof is prepared by the purification method of the invention; the polymer is preferably a hydrophilic polymer including, but not limited to: polysaccharides, polypeptides, polyalkylene glycols, polyvinylpyrrolidone, polyacrylates, polymethacrylates, polyoxazolines, polyvinyl alcohols, polyacrylamides, polymethacrylamides, maleic acid acrylic copolymers, polyesters, polyacetals, polyorthoesters, polycarbonates, polyurethane carbonates, polyamides, vinyl ether maleic anhydride copolymers, styrene maleic anhydride copolymers and copolymers of the above polymers, more preferably polyethylene glycol.

The polyethylene glycol is referred to as "PEG", specifically, a mixture of a polycondensate of ethylene oxide and water, represented by the general formula H (OCH ₂CH₂)_n OH, where n.gtoreq.4. Generally, the molecular weight of the PEG molecule is 5kDa, and these PEG may be a linear chain or a branched chain.

Taking the example of a conjugate of uricase or an isoform thereof and polyethylene glycol, uricase or an isoform thereof may be covalently bonded to polyethylene glycol (PEG) via chemical bonds using methods known in the art, without departing from the ability of those skilled in the art. Typically, PEG can be conjugated to mutant uricase via a linking group, wherein the linking group can be selected from the group consisting of: succinimidyl, amido, imidyl, carbamate, ester, epoxy, carboxyl, hydroxyl, carbohydrate, tyrosine, cysteine, histidine groups, or combinations thereof. Alternatively, uricase or an isoform thereof may be coupled directly to PEG (i.e., without a linking group) via an amino group, a thiol group, a hydroxyl group, or a carboxyl group.

As an example, as described in WO 2000007629:

Different molecular weight (5-30 kDa) monomethoxy PEG derivatives, such as 4-nitrophenylcarbonate PEG (NPC-PEG), were added to 0.1M, pH 10.2.2 sodium carbonate buffer in which tetrameric urate oxidase was dissolved, in a ratio of 10-200 moles of activated PEG per mole of urate oxidase subunit (molecular weight 35 kDa). This and other suitable activated PEGs are commercially available from SHEARWATER POLYMERS. The instructions for the coupling reaction of these PEG with proteins are given in the SHEARWATER POLYMERS list on the Internet www.swpolymers.com and JM Harris,et al.,(Eds.)(1997)Poly(ethylene glycol)Chemistry and BiologicalApplications.ACS Symposium Series 680,Washington,DC：AmericanChemical Society. at a temperature of 0-8deg.C until the degree of PEG coupling is no longer time-dependent. Unreacted PEG is then removed from the reaction product by chromatography and/or ultrafiltration.

The number of PEG chains coupled per urate oxidase subunit was determined by Kunitani, M, et al, (1991) J Chromatogr 588:125-137; saifer, et al, (1997) and Sherman, et al, (1997) as determined after modification of the method. Briefly, the components of the PEGylation reaction mixture or the fraction eluted from the preparative ion exchange or size separation column are identified by size separation high pressure liquid chromatography performed at room temperature, suitably a TSK 5,000PWXL column. The buffer was a sodium carbonate solution containing 0.1M NaCl at pH 10.2. The high performance liquid chromatography column was purchased from TosoHaas, montgomeryville, PA. Proteins and PEG were detected by measuring uv absorbance and using a refractive index detector. The protein content of the conjugate was calculated by uv absorbance and used as a reference with a standard solution of the appropriate unmodified urate oxidase. The amount of PEG in the conjugate was calculated from the area of the refractive index peak, and the result was referenced to the refractive index peak area of the appropriate PEG standard solution, with the final result being a corrected value taking into account the effect of the protein on refractive index.

The invention further provides a preparation method of a composition containing uricase or a conjugate of the uricase isoform, which comprises the steps of conjugation of the uricase or the uricase isoform with a polymer and mixing with pharmaceutically acceptable auxiliary materials, wherein the uricase or the uricase isoform is prepared by the purification method; the optional polymer species is preferably polyethylene glycol as previously described.

Based on the research, the invention also provides application of the uricase or the isoform thereof in preparing medicines for treating diseases caused by elevated uric acid; the disease includes one or more of the diseases including metabolic syndrome, hyperuricemia, gout such as gouty arthritis, lesch-Nyhan syndrome, cardiovascular disease, diabetes, hypertension, kidney disease, uric acid kidney stones or kidney stones, oncolytic syndrome or hyperuricuria; preferably, the disease comprises hyperuricemia and/or gout.

More importantly, the invention also provides a medicine which comprises the uricase or the isoform thereof and pharmaceutically acceptable auxiliary materials or carriers. The drug may be administered by injection by intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal routes or by enteral administration through an oral dosage form. Elevated uric acid levels may be present in blood or urine and may be associated with gout, tophaceous, renal insufficiency, organ transplantation, or malignant disease.

"Uricase" according to the invention, unless specified otherwise, refers to wild-type uricase, i.e., a naturally occurring form within the species, which uricase may be derived from humans, mammals, plants, microorganisms, preferably from microorganisms, more preferably from fungi and bacteria, such as, for example, arthrobacter sphaeroides, aspergillus flavus, candida utilis, bacillus fastidious, etc., most preferably from Arthrobacter sphaeroides (e.g., SEQ ID NO. 1);

the term "isoforms of uricase" as used herein refers to mutants, fragments, isomers, derivatives, chimeras or combinations thereof of wild-type uricase activity, unless specifically indicated.

Mutants of uricase refer to uricase molecules obtained by amino acid substitution, deletion, addition, and in some embodiments, preferably uricase having at most 10 amino acid substitutions, deletions, additions, more preferably uricase having at most 5 amino acid substitutions, deletions, additions, still more preferably uricase having at most 2 amino acid substitutions, deletions, additions, and even more preferably uricase having at most 1 amino acid substitution, deletion, addition, as compared to wild type uricase;

In other embodiments, mutants of uricase have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity compared to wild type uricase; sequence alignment methods for comparison are well known in the art and suitable algorithms for determining percent sequence identity include, for example, BLAST and BLAST2.0 algorithms (see Altschul et al, nuc. Acids Res.25:3389-402,1977 and Altschul et al J. Mol. Biol.215:403-10,1990. Software available through the national center of Biotechnology information (National Center for Biotechnology Information) public acquisition (http:// www.ncbi.nlm.nih.gov /) for BLAST analysis for purposes of the present application, percent identity is generally determined using the BLAST2.0 algorithm set as a default parameter.

It will be appreciated by those skilled in the art that uricase mutants obtained by conservative substitutions, meaning amino acid substitutions that do not adversely affect or alter the biological function of the protein/polypeptide comprising the amino acid sequence, are within the scope of the invention. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions refer to the substitution of one amino acid for another that has similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that can be typically conservatively substituted with each other: 1) Alanine (a), serine (S), threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q);

4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

Fragments of uricase refer to any smaller portion of the sequence that belongs to wild-type uricase, and may be, but are not limited to, obtained by cleavage, or expressed genetically, or obtained by methods of polypeptide synthesis; an isomer of uricase refers to an enzyme having the same amino acid sequence or molecular formula as wild-type uricase, but having a different conformation, including a difference in secondary or tertiary structure of the protein or a change in optical activity of a local amino acid, and may be naturally occurring or artificially designed; the derivative of uricase refers to a product modified on the basis of wild uricase, wherein the modification refers to covalent linkage of one or more other small molecules, such as phosphoric acid molecules, sugar molecules and the like, or a small peptide chain with less than 20 amino acids, and the linkage site can be any amino acid in the protein; chimeras of uricase refer to chimeric products of wild type uricase derived from different species, including, but not limited to, human-canine chimeric uricase, human-porcine chimeric uricase, human-baboon chimeric uricase, and the like; a combination of mutants, fragments, isomers, derivatives, chimeras of uricase activity refers to products having two or more changes in the above characteristics at the same time, such as, but not limited to, mutants of fragments, modified products of mutants, and the like.

By "active" is meant that the mutant, fragment, isomer, derivative, chimera or combination thereof retains more than 50% of the enzymatic activity, preferably more than 60% of the enzymatic activity, more preferably more than 70% of the enzymatic activity, even more preferably more than 80% of the enzymatic activity of the wild-type uricase.

In a specific embodiment of the present invention, uricase (SEQ ID NO. 1) derived from Arthrobacter sphaeroides (Arthrobacter globiformils) having high activity and low immunogenicity, hereinafter referred to as AgU, is selected.

As a series of examples of uricase mutants, site-directed mutagenesis was performed on the basis of native uricase AgU for Lysine (K) residues that may be the site of PEG modification to reduce the number of PEG modifications and preserve as much of the enzyme activity as possible. The amino acid sequence of natural uricase is shown in protein sequence 1 (SEQ ID No. 1), and the site-directed mutation sites are respectively changed from lysine 29 to arginine (AgU K R, SEQ ID No. 2), from lysine 71 to arginine (AgU K R, SEQ ID No. 3), from lysine 94 to arginine (AgU K R, SEQ ID No. 4), from lysine 170 to arginine (AgU K R, SEQ ID No. 5), from lysine 212 to arginine (AgU K R, SEQ ID No. 6), from lysine 244 to arginine (AgU K R, SEQ ID No. 7) and from lysine 250 to arginine (AgU K250R, SEQ ID No. 8).

Uricase or an isoform thereof to be purified herein may be obtained by methods known in the art, including but not limited to isolation from nature, chemical synthesis, or expression by genetic engineering, and in one embodiment the invention employs E.coli to express the uricase followed by protein purification. In the purification process, the inventor finds that the purity of uricase or an isoform thereof obtained by two-step purification can reach more than 95 percent through hydrophobic chromatography and anion exchange chromatography purification, the protein recovery rate is close to 50 percent, and the specific activity of uricase is high. Compared with the prior art, the uricase or the isoform thereof obtained by the preparation method has obviously improved purity and specific activity, and simultaneously has high protein recovery rate, thereby being suitable for industrial production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a diagram of RP-HPLC results for determining the purity of a target protein;

FIG. 2 shows SDS-PAGE electrophoresis for determining the purity of the target protein.

Detailed Description

The invention discloses uricase or an isoform thereof and a preparation method thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

It should be understood that the expression "one or more of … …" individually includes each stated object after the expression and various combinations of two or more of the stated objects unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood as having the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, should generally be construed as open-ended and non-limiting, e.g., not to exclude other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.

It should be understood that the order of steps or order of performing certain actions is not important so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language, such as "e.g." or "comprising" herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Furthermore, the numerical ranges and parameters setting forth the present invention are approximations that may vary as precisely as possible in the exemplary embodiments. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used in this disclosure are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.

In the uricase or the isoform thereof provided by the invention and the preparation method thereof, raw materials and reagents used in the uricase or the isoform thereof can be purchased from the market.

The invention is further illustrated by the following examples:

example 1: construction of uricase-expressing Strain

According to the protein sequences 1-8 (SEQ ID No.1-SEQ ID No. 8), the Nanjing Jinsri Biotechnology Co.Ltd was delegated to complete gene synthesis and inserted into the vector pET28a (+) in the middle of two cleavage sites NcoI and XhoI. The gene was optimized according to the preferred codons of E.coli, and its sequence was gene sequences 9-16 (SEQ ID No.9-SEQ ID No. 16), respectively.

SEQ ID No.1

MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC

SEQ ID No.2

MTATAETSTGTKVVLGQNQYGKAEVRLVRVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC

SEQ ID No.3

MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQRNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC

SEQ ID No.4

MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGRHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC

SEQ ID No.5

MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDRYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC

SEQ ID No.6

MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLRAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC

SEQ ID No.7

MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIRMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC

SEQ ID No.8

MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNRHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC

SEQ ID No.9

ATGACCGCGACCGCGGAAACCAGCACCGGTACCAAGGTGGTTCTGGGTCAGAACCAATACGGCAAGGCGGAAGTGCGTCTGGTGAAAGTTACCCGTAACACCGCGCGTCACGAAATCCAGGACCTGAACGTTACCAGCCAACTGCGTGGTGATTTTGAGGCGGCGCATACCGCGGGTGACAACGCGCACAAGGTGGCGACCGATACCCAGAAAAACACCGTTTATGCGTTCGCGCGTGACGGTTTTGCGACCACCGAGGAATTTCTGCTGCGTCTGGGCAAGCACTTCACCGAAGGCTTTGACTGGGTGACCGGTGGCCGTTGGGCGGCGCAGCAATTCTTTTGGGATCGTATCAACGACCACGATCACGCGTTCAGCCGTAACAAAAGCGAGGTGCGCACCGCGGTTCTGGAGATTAGCGGCAGCGAACAGGCGATCGTGGCGGGTATTGAGGGCCTGACCGTTCTGAAGAGCACCGGTAGCGAATTCCACGGCTTTCCGCGTGACAAATACACCACCCTGCAAGAGACCACCGACCGTATTCTGGCGACCGATGTGAGCGCGCGTTGGCGTTACAACACCGTGGAAGTTGACTTCGATGCGGTGTATGCGAGCGTTCGTGGTCTGCTGCTGAAAGCGTTTGCGGAGACCCACAGCCTGGCGCTGCAGCAAACCATGTATGAGATGGGCCGTGCGGTTATTGAAACCCACCCGGAGATCGATGAAATTAAGATGAGCCTGCCGAACAAACACCACTTCCTGGTGGACCTGCAGCCGTTTGGTCAAGATAACCCGAACGAAGTTTTCTACGCGGCGGACCGTCCGTATGGTCTGATCGAGGCGACCATTCAACGTGAAGGCAGCCGTGCGGATCACCCGATCTGGAGCAACATTGCGGGCTTTTGCTAA

SEQ ID No.10ATGACCGCGACCGCGGAAACCAGCACCGGTACCAAGGTGGTTCTGGGTCAGAACCAATACGGCAAAGCGGAAGTGCGTCTGGTGCGTGTTACCCGTAACACCGCGCGTCACGAAATCCAGGACCTGAACGTTACCAGCCAACTGCGTGGTGATTTTGAGGCGGCGCATACCGCGGGTGACAACGCGCATGTGGTTGCGACCGATACCCAGAAGAACACCGTGTATGCGTTCGCGCGTGACGGTTTTGCGACCACCGAGGAATTTCTGCTGCGTCTGGGTAAACACTTCACCGAAGGCTTTGACTGGGTTACCGGTGGCCGTTGGGCGGCGCAGCAATTCTTTTGGGATCGTATCAACGACCACGATCACGCGTTCAGCCGTAACAAGAGCGAGGTGCGCACCGCGGTTCTGGAGATTAGCGGCAGCGAACAGGCGATCGTGGCGGGTATTGAGGGCCTGACCGTTCTGAAGAGCACCGGTAGCGAATTCCACGGCTTTCCGCGTGACAAATACACCACCCTGCAAGAGACCACCGACCGTATTCTGGCGACCGATGTGAGCGCGCGTTGGCGTTACAACACCGTGGAAGTTGACTTCGATGCGGTGTATGCGAGCGTTCGTGGTCTGCTGCTGAAAGCGTTTGCGGAGACCCACAGCCTGGCGCTGCAGCAAACCATGTATGAGATGGGCCGTGCGGTTATTGAAACCCACCCGGAGATCGATGAAATTAAGATGAGCCTGCCGAACAAACACCACTTCCTGGTGGACCTGCAGCCGTTTGGTCAAGATAACCCGAACGAAGTTTTCTACGCGGCGGACCGTCCGTATGGTCTGATCGAGGCGACCATTCAACGTGAAGGCAGCCGTGCGGATCACCCGATCTGGAGCAACATTGCGGGCTTTTGCTAA

SEQ ID No.11

ATGACCGCGACCGCGGAAACCAGCACCGGTACCAAGGTGGTTCTGGGTCAGAACCAATACGGCAAGGCGGAAGTGCGTCTGGTGAAAGTTACCCGTAACACCGCGCGTCACGAAATCCAGGACCTGAACGTTACCAGCCAACTGCGTGGTGATTTTGAGGCGGCGCATACCGCGGGTGACAACGCGCATGTGGTTGCGACCGATACCCAGCGTAACACCGTGTATGCGTTCGCGCGTGACGGTTTTGCGACCACCGAGGAATTTCTGCTGCGTCTGGGCAAGCACTTCACCGAAGGCTTTGACTGGGTTACCGGTGGCCGTTGGGCGGCGCAGCAATTCTTTTGGGATCGTATCAACGACCACGATCACGCGTTCAGCCGTAACAAAAGCGAGGTGCGCACCGCGGTTCTGGAGATTAGCGGCAGCGAACAGGCGATCGTGGCGGGTATTGAGGGCCTGACCGTTCTGAAGAGCACCGGTAGCGAATTCCACGGCTTTCCGCGTGACAAATACACCACCCTGCAAGAGACCACCGACCGTATTCTGGCGACCGATGTGAGCGCGCGTTGGCGTTACAACACCGTGGAAGTTGACTTCGATGCGGTGTATGCGAGCGTTCGTGGTCTGCTGCTGAAAGCGTTTGCGGAGACCCACAGCCTGGCGCTGCAGCAAACCATGTATGAGATGGGCCGTGCGGTTATTGAAACCCACCCGGAGATCGATGAAATTAAGATGAGCCTGCCGAACAAACACCACTTCCTGGTGGACCTGCAGCCGTTTGGTCAAGATAACCCGAACGAAGTTTTCTACGCGGCGGACCGTCCGTATGGTCTGATCGAGGCGACCATTCAACGTGAAGGCAGCCGTGCGGATCACCCGATCTGGAGCAACATTGCGGGCTTTTGCTAA

SEQ ID No.12

ATGACCGCGACCGCGGAAACCAGCACCGGTACCAAGGTGGTTCTGGGTCAGAACCAATACGGCAAGGCGGAAGTGCGTCTGGTGAAAGTTACCCGTAACACCGCGCGTCACGAAATCCAGGACCTGAACGTTACCAGCCAACTGCGTGGTGATTTTGAGGCGGCGCATACCGCGGGTGACAACGCGCATGTGGTTGCGACCGATACCCAGAAGAACACCGTGTATGCGTTCGCGCGTGACGGTTTTGCGACCACCGAGGAATTTCTGCTGCGTCTGGGTCGTCACTTCACCGAAGGCTTTGACTGGGTTACCGGTGGCCGTTGGGCGGCGCAGCAATTCTTTTGGGATCGTATCAACGACCACGATCACGCGTTCAGCCGTAACAAAAGCGAGGTGCGCACCGCGGTTCTGGAGATTAGCGGCAGCGAACAGGCGATCGTGGCGGGTATTGAGGGCCTGACCGTTCTGAAGAGCACCGGTAGCGAATTCCACGGCTTTCCGCGTGACAAATACACCACCCTGCAAGAGACCACCGACCGTATTCTGGCGACCGATGTGAGCGCGCGTTGGCGTTACAACACCGTGGAAGTTGACTTCGATGCGGTGTATGCGAGCGTTCGTGGTCTGCTGCTGAAAGCGTTTGCGGAGACCCACAGCCTGGCGCTGCAGCAAACCATGTATGAGATGGGCCGTGCGGTTATTGAAACCCACCCGGAGATCGATGAAATTAAGATGAGCCTGCCGAACAAACACCACTTCCTGGTGGACCTGCAGCCGTTTGGTCAAGATAACCCGAACGAAGTTTTCTACGCGGCGGACCGTCCGTATGGTCTGATCGAGGCGACCATTCAACGTGAAGGCAGCCGTGCGGATCACCCGATCTGGAGCAACATTGCGGGCTTTTGCTAA

SEQ ID No.13

ATGACCGCGACCGCGGAAACCAGCACCGGTACCAAGGTGGTTCTGGGTCAGAACCAATACGGCAAGGCGGAAGTGCGTCTGGTGAAAGTTACCCGTAACACCGCGCGTCACGAAATCCAGGACCTGAACGTTACCAGCCAACTGCGTGGTGATTTTGAGGCGGCGCATACCGCGGGTGACAACGCGCATGTGGTTGCGACCGATACCCAGAAGAACACCGTGTATGCGTTCGCGCGTGACGGTTTTGCGACCACCGAGGAATTTCTGCTGCGTCTGGGTAAACACTTCACCGAAGGCTTTGACTGGGTTACCGGTGGCCGTTGGGCGGCGCAGCAATTCTTTTGGGATCGTATCAACGACCACGATCACGCGTTCAGCCGTAACAAGAGCGAGGTGCGCACCGCGGTTCTGGAGATTAGCGGCAGCGAACAGGCGATCGTGGCGGGTATTGAGGGCCTGACCGTTCTGAAAAGCACCGGTAGCGAATTCCACGGCTTTCCGCGTGACCGTTACACCACCCTGCAAGAGACCACCGACCGTATTCTGGCGACCGATGTGAGCGCGCGTTGGCGTTACAACACCGTGGAAGTTGACTTCGATGCGGTGTATGCGAGCGTTCGTGGTCTGCTGCTGAAAGCGTTTGCGGAGACCCACAGCCTGGCGCTGCAGCAAACCATGTATGAGATGGGCCGTGCGGTTATTGAAACCCACCCGGAGATCGATGAAATTAAGATGAGCCTGCCGAACAAACACCACTTCCTGGTGGACCTGCAGCCGTTTGGTCAAGATAACCCGAACGAAGTTTTCTACGCGGCGGACCGTCCGTATGGTCTGATCGAGGCGACCATTCAACGTGAAGGCAGCCGTGCGGATCACCCGATCTGGAGCAACATTGCGGGCTTTTGCTAA

SEQ ID No.14

ATGACCGCGACCGCGGAAACCAGCACCGGTACCAAGGTGGTTCTGGGTCAGAACCAATACGGCAAGGCGGAAGTGCGTCTGGTGAAAGTTACCCGTAACACCGCGCGTCACGAAATCCAGGACCTGAACGTTACCAGCCAACTGCGTGGTGATTTTGAGGCGGCGCATACCGCGGGTGACAACGCGCATGTGGTTGCGACCGATACCCAGAAGAACACCGTGTATGCGTTCGCGCGTGACGGTTTTGCGACCACCGAGGAATTTCTGCTGCGTCTGGGTAAACACTTCACCGAAGGCTTTGACTGGGTTACCGGTGGCCGTTGGGCGGCGCAGCAATTCTTTTGGGATCGTATCAACGACCACGATCACGCGTTCAGCCGTAACAAAAGCGAGGTGCGCACCGCGGTTCTGGAGATTAGCGGCAGCGAACAGGCGATCGTGGCGGGTATTGAGGGCCTGACCGTTCTGAAGAGCACCGGTAGCGAATTCCACGGCTTTCCGCGTGACAAATACACCACCCTGCAAGAGACCACCGACCGTATTCTGGCGACCGATGTGAGCGCGCGTTGGCGTTACAACACCGTGGAAGTTGACTTCGATGCGGTGTATGCGAGCGTTCGTGGTCTGCTGCTGCGTGCGTTTGCGGAGACCCACAGCCTGGCGCTGCAGCAAACCATGTATGAGATGGGCCGTGCGGTTATTGAAACCCACCCGGAGATCGATGAAATTAAGATGAGCCTGCCGAACAAACACCACTTCCTGGTGGACCTGCAGCCGTTTGGTCAAGATAACCCGAACGAAGTTTTCTACGCGGCGGACCGTCCGTATGGTCTGATCGAGGCGACCATTCAACGTGAAGGCAGCCGTGCGGATCACCCGATCTGGAGCAACATTGCGGGCTTTTGCTAA

SEQ ID No.15

ATGACCGCGACCGCGGAAACCAGCACCGGTACCAAGGTGGTTCTGGGTCAGAACCAATACGGCAAGGCGGAAGTGCGTCTGGTGAAAGTTACCCGTAACACCGCGCGTCACGAAATCCAGGACCTGAACGTTACCAGCCAACTGCGTGGTGATTTTGAGGCGGCGCATACCGCGGGTGACAACGCGCATGTGGTTGCGACCGATACCCAGAAGAACACCGTGTATGCGTTCGCGCGTGACGGTTTTGCGACCACCGAGGAATTTCTGCTGCGTCTGGGTAAACACTTCACCGAAGGCTTTGACTGGGTTACCGGTGGCCGTTGGGCGGCGCAGCAATTCTTTTGGGATCGTATCAACGACCACGATCACGCGTTCAGCCGTAACAAAAGCGAGGTGCGCACCGCGGTTCTGGAGATTAGCGGCAGCGAACAGGCGATCGTGGCGGGTATTGAGGGCCTGACCGTTCTGAAGAGCACCGGTAGCGAATTCCACGGCTTTCCGCGTGACAAATACACCACCCTGCAAGAGACCACCGACCGTATTCTGGCGACCGATGTGAGCGCGCGTTGGCGTTACAACACCGTGGAAGTTGACTTCGATGCGGTGTATGCGAGCGTTCGTGGTCTGCTGCTGAAGGCGTTTGCGGAGACCCACAGCCTGGCGCTGCAGCAAACCATGTATGAGATGGGCCGTGCGGTTATTGAAACCCACCCGGAGATCGATGAAATTCGTATGAGCCTGCCGAACAAACACCACTTCCTGGTGGACCTGCAGCCGTTTGGTCAAGATAACCCGAACGAAGTTTTCTACGCGGCGGACCGTCCGTATGGTCTGATCGAGGCGACCATTCAACGTGAAGGCAGCCGTGCGGATCACCCGATCTGGAGCAACATTGCGGGCTTTTGCTAA

SEQ ID No.16

ATGACCGCGACCGCGGAAACCAGCACCGGTACCAAGGTGGTTCTGGGTCAGAACCAATACGGCAAGGCGGAAGTGCGTCTGGTGAAAGTTACCCGTAACACCGCGCGTCACGAAATCCAGGACCTGAACGTTACCAGCCAACTGCGTGGTGATTTTGAGGCGGCGCATACCGCGGGTGACAACGCGCATGTGGTTGCGACCGATACCCAGAAGAACACCGTGTATGCGTTCGCGCGTGACGGTTTTGCGACCACCGAGGAATTTCTGCTGCGTCTGGGTAAACACTTCACCGAAGGCTTTGACTGGGTTACCGGTGGCCGTTGGGCGGCGCAGCAATTCTTTTGGGATCGTATCAACGACCACGATCACGCGTTCAGCCGTAACAAAAGCGAGGTGCGCACCGCGGTTCTGGAGATTAGCGGCAGCGAACAGGCGATCGTGGCGGGTATTGAGGGCCTGACCGTTCTGAAGAGCACCGGTAGCGAATTCCACGGCTTTCCGCGTGACAAATACACCACCCTGCAAGAGACCACCGACCGTATTCTGGCGACCGATGTGAGCGCGCGTTGGCGTTACAACACCGTGGAAGTTGACTTCGATGCGGTGTATGCGAGCGTTCGTGGTCTGCTGCTGAAGGCGTTTGCGGAGACCCACAGCCTGGCGCTGCAGCAAACCATGTATGAGATGGGCCGTGCGGTTATTGAAACCCACCCGGAGATCGATGAAATTAAAATGAGCCTGCCGAACCGTCACCACTTCCTGGTGGACCTGCAGCCGTTTGGTCAAGATAACCCGAACGAAGTTTTCTACGCGGCGGACCGTCCGTATGGTCTGATCGAGGCGACCATTCAACGTGAAGGCAGCCGTGCGGATCACCCGATCTGGAGCAACATTGCGGGCTTTTGCTAA

After complete gene synthesis, it was transferred into E.coli expression host BL21 (DE 3). The method comprises the following specific steps: melting BL21 (DE 3) competent cells in ice bath, adding target DNA, and standing in ice bath for 30min; the centrifuge tube was placed in a 42 ℃ water bath for 90sec, then the tube was quickly transferred to an ice bath and the cells were allowed to cool for 3min. Sterile LB medium (without antibiotics) was added to the tube, mixed well and placed in shaking table at 37℃for 45min (150 rpm). The centrifuge tube contents were mixed and 100. Mu.l of transformed competent cells were pipetted onto LB solid agar medium containing kanamycin and gently spread out using a sterile spreading bar. The plate is placed at room temperature until liquid is absorbed, the plate is inverted, static culture is carried out at 37 ℃ for overnight, single bacterial colony is picked up, inoculated in 1ml of liquid LB culture medium containing kanamycin (50 ug/ml), shake culture is carried out at 37 ℃ for overnight, the cultured bacterial liquid is inoculated in 10ml of liquid LB culture medium containing kanamycin (50 ug/ml), culture is carried out at 37 ℃ for 4 hours at 250rpm, 5ml of bacterial liquid is added into 0.1mM IPTG for induction overnight, SDS-PAGE detection induction condition is carried out, bacterial bodies which induce target proteins are selected, and other 5ml bacterial liquid which is not added with IPTG induction is added into glycerol for freezing storage at-80 ℃.

Example 2: uricase expression and purification

1. Protein expression

1. Resuscitates the target strain: taking out a glycerol frozen strain stored at-80 ℃, inoculating the bacterial liquid into LB culture medium containing kanamycin (50 ug/mL) according to the ratio of 1:100, and shaking overnight culturing in a shaking table at 37 ℃ and 250 rpm.

2. Enlarging and inducing target strain: resuscitator solution was inoculated at a ratio of 1:100 into LB medium containing kanamycin (50 ug/mL) and cultured overnight with shaking in a shaker at 37℃at 250 rpm. When the OD600 is 0.6-0.8, 0.1mM IPTG is added to induce expression, and the culture is carried out for 16h in a shaking table at 37 ℃ and 250 rpm.

2. Cell disruption

The culture broth was centrifuged at 10000rpm at 4℃for 10min, the precipitated cells were collected, washed with 50mM borax borate buffer at pH=8.5, centrifuged at 10000rpm at 4℃for 10min, the precipitated cells were collected, resuspended in 100ml of a disruption buffer (borax borate at 50mM pH 8.5), sonicated in ice bath (ultrasonic power 50% for 30 min), and centrifuged at 10000rpm at 4℃for 30min, and the supernatant was collected.

3. Protein purification

1. Sample treatment: the crushed supernatant was added (NH ₄)₂SO₄ to a final concentration of 1M, and after mixing, the supernatant was collected after centrifugation at 10000rpm for 10min at 4 ℃.

HIC column separation and purification:

Loading: adding 100ml of treated sample, and loading at 5ml/min

3. Sample treatment: the eluted proteins in step 2 were diluted 10-fold with 50mM borax borate buffer pH 8.5.

4. Anion exchange column chromatography (Anion-IEX) separation:

And (3) purification:

4. Determination of recovery of target protein

Determining the total enzyme activity of the bacterial liquid disruption supernatant and the total enzyme activity after purification AgU according to the enzyme activity detection method disclosed in example 3; as a result, the total enzyme activity of the supernatant obtained by disruption of the AgU strain was 2831.32IU, the total enzyme activity after purification was 1311.24IU, and the recovery rate of the latter was 46.31% as compared with the former.

5. Determination of the purity of the protein of interest

5.1RP-HPLC determination of the purity of the protein of interest

The purity of AgU after purification was determined by RP-HPLC and the results are shown in FIG. 1 and Table 1, the chromatographic conditions are as follows:

Chromatographic column: jupiter 5. Mu.mC 18

Mobile phase a:5% acetonitrile +0.1% trifluoroacetic acid

Mobile phase B:95% acetonitrile +0.1 trifluoroacetic acid

Flow rate: 1.0ml/min

Detection wavelength: 214nm

The gradient method comprises the following steps: the linear gradient is 30min, and the liquid B is 0-100%.

TABLE 1

Sample name	Purity of	Impurity(s)
			AgU	99.53％	0.47％

5.2SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) for determining the purity of the target protein

AgU and allosteric proteins were obtained according to the above uricase expression and purification method, 12% SDS-PAGE was followed by coomassie brilliant blue staining, and the gray scale ratio of the quality one software was used to analyze the gray scale value of each allosteric protein as the percentage (%) of the gray scale value of each total allosteric protein as the purity, and the results are shown in Table 2.

TABLE 2

Uricase abbreviation	Total gray scale	Target protein gray scale	Purity (%)
				AgU	39674.11	39359.64	99.71
AgU29R	36435.85	32584.58	89.43
				AgU94R	95151.62	8217.69	86.36
AgU244R	40709.55	38006.44	93.36
				AgU250R	40126.50	37787.13	94.17

Example 3: enzymatic detection of uricase

Urate oxidase catalyzes uric acid to catalyze uric acid to decompose, and uric acid has a characteristic absorption peak at 290 nm. However, the allantoin, which is the product of uric acid decomposition, has no absorption peak at this wavelength, so that the amount of uric acid degraded by urate oxidase can be determined from the decrease in absorption light at 290nm, and then the uric acid concentration is calculated from the uric acid standard curve, and the uricase activity is calculated from the amount of uric acid consumed. The wavelength of the enzyme label instrument is adjusted to 290nm, the temperature is adjusted to 37 ℃, and the instrument is preheated. 0.1ml of 0.6mmol/L uric acid solution was taken into a 96-well UV-UV transparent flat bottom plate. Preheating at 37 deg.c for 10min. Adding 0.1ml diluted enzyme solution, quickly mixing, reading, recording absorbance value every 5min, and measuring the change value of absorbance value at 290nm within 1 h.

The amount of enzyme that converts 1. Mu. Mol uric acid to allantoin per minute at 37℃and pH8.5 was determined as one International Unit (IU). And (3) making a uric acid standard curve according to the OD290 reading value, substituting the measured OD290 absorbance value of the sample into a formula to calculate the residual uric acid concentration C ₁. The recombinant uricase activity was then calculated according to the following formula:

Enzyme specific activity (U/mg) = (U/ml) ×1/C2

C2: enzyme concentration (mg/ml), t: reaction time

The results were as follows:

TABLE 3 specific Activity after separation and purification of uricase and allosteric protein

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Paiger biomedical (Suzhou) stock Co., ltd

<120> Uricase or an isoform thereof and methods of making the same

<130> MP21005213

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 302

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 2

<211> 302

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Arg Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 3

<211> 302

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Arg Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 4

<211> 302

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Arg His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 5

<211> 302

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Arg Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 6

<211> 302

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Arg Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 7

<211> 302

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Arg Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 8

<211> 302

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Arg His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 9

<211> 909

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

atgaccgcga ccgcggaaac cagcaccggt accaaggtgg ttctgggtca gaaccaatac 60

ggcaaggcgg aagtgcgtct ggtgaaagtt acccgtaaca ccgcgcgtca cgaaatccag 120

gacctgaacg ttaccagcca actgcgtggt gattttgagg cggcgcatac cgcgggtgac 180

aacgcgcaca aggtggcgac cgatacccag aaaaacaccg tttatgcgtt cgcgcgtgac 240

ggttttgcga ccaccgagga atttctgctg cgtctgggca agcacttcac cgaaggcttt 300

gactgggtga ccggtggccg ttgggcggcg cagcaattct tttgggatcg tatcaacgac 360

cacgatcacg cgttcagccg taacaaaagc gaggtgcgca ccgcggttct ggagattagc 420

ggcagcgaac aggcgatcgt ggcgggtatt gagggcctga ccgttctgaa gagcaccggt 480

agcgaattcc acggctttcc gcgtgacaaa tacaccaccc tgcaagagac caccgaccgt 540

attctggcga ccgatgtgag cgcgcgttgg cgttacaaca ccgtggaagt tgacttcgat 600

gcggtgtatg cgagcgttcg tggtctgctg ctgaaagcgt ttgcggagac ccacagcctg 660

gcgctgcagc aaaccatgta tgagatgggc cgtgcggtta ttgaaaccca cccggagatc 720

gatgaaatta agatgagcct gccgaacaaa caccacttcc tggtggacct gcagccgttt 780

ggtcaagata acccgaacga agttttctac gcggcggacc gtccgtatgg tctgatcgag 840

gcgaccattc aacgtgaagg cagccgtgcg gatcacccga tctggagcaa cattgcgggc 900

ttttgctaa 909

<210> 10

<211> 909

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

atgaccgcga ccgcggaaac cagcaccggt accaaggtgg ttctgggtca gaaccaatac 60

ggcaaagcgg aagtgcgtct ggtgcgtgtt acccgtaaca ccgcgcgtca cgaaatccag 120

gacctgaacg ttaccagcca actgcgtggt gattttgagg cggcgcatac cgcgggtgac 180

aacgcgcatg tggttgcgac cgatacccag aagaacaccg tgtatgcgtt cgcgcgtgac 240

ggttttgcga ccaccgagga atttctgctg cgtctgggta aacacttcac cgaaggcttt 300

gactgggtta ccggtggccg ttgggcggcg cagcaattct tttgggatcg tatcaacgac 360

cacgatcacg cgttcagccg taacaagagc gaggtgcgca ccgcggttct ggagattagc 420

ggcagcgaac aggcgatcgt ggcgggtatt gagggcctga ccgttctgaa gagcaccggt 480

agcgaattcc acggctttcc gcgtgacaaa tacaccaccc tgcaagagac caccgaccgt 540

attctggcga ccgatgtgag cgcgcgttgg cgttacaaca ccgtggaagt tgacttcgat 600

gcggtgtatg cgagcgttcg tggtctgctg ctgaaagcgt ttgcggagac ccacagcctg 660

gcgctgcagc aaaccatgta tgagatgggc cgtgcggtta ttgaaaccca cccggagatc 720

gatgaaatta agatgagcct gccgaacaaa caccacttcc tggtggacct gcagccgttt 780

ggtcaagata acccgaacga agttttctac gcggcggacc gtccgtatgg tctgatcgag 840

gcgaccattc aacgtgaagg cagccgtgcg gatcacccga tctggagcaa cattgcgggc 900

ttttgctaa 909

<210> 11

<211> 909

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

atgaccgcga ccgcggaaac cagcaccggt accaaggtgg ttctgggtca gaaccaatac 60

ggcaaggcgg aagtgcgtct ggtgaaagtt acccgtaaca ccgcgcgtca cgaaatccag 120

gacctgaacg ttaccagcca actgcgtggt gattttgagg cggcgcatac cgcgggtgac 180

aacgcgcatg tggttgcgac cgatacccag cgtaacaccg tgtatgcgtt cgcgcgtgac 240

ggttttgcga ccaccgagga atttctgctg cgtctgggca agcacttcac cgaaggcttt 300

gactgggtta ccggtggccg ttgggcggcg cagcaattct tttgggatcg tatcaacgac 360

cacgatcacg cgttcagccg taacaaaagc gaggtgcgca ccgcggttct ggagattagc 420

ggcagcgaac aggcgatcgt ggcgggtatt gagggcctga ccgttctgaa gagcaccggt 480

agcgaattcc acggctttcc gcgtgacaaa tacaccaccc tgcaagagac caccgaccgt 540

attctggcga ccgatgtgag cgcgcgttgg cgttacaaca ccgtggaagt tgacttcgat 600

gcggtgtatg cgagcgttcg tggtctgctg ctgaaagcgt ttgcggagac ccacagcctg 660

gcgctgcagc aaaccatgta tgagatgggc cgtgcggtta ttgaaaccca cccggagatc 720

gatgaaatta agatgagcct gccgaacaaa caccacttcc tggtggacct gcagccgttt 780

ggtcaagata acccgaacga agttttctac gcggcggacc gtccgtatgg tctgatcgag 840

gcgaccattc aacgtgaagg cagccgtgcg gatcacccga tctggagcaa cattgcgggc 900

ttttgctaa 909

<210> 12

<211> 909

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

atgaccgcga ccgcggaaac cagcaccggt accaaggtgg ttctgggtca gaaccaatac 60

ggcaaggcgg aagtgcgtct ggtgaaagtt acccgtaaca ccgcgcgtca cgaaatccag 120

gacctgaacg ttaccagcca actgcgtggt gattttgagg cggcgcatac cgcgggtgac 180

aacgcgcatg tggttgcgac cgatacccag aagaacaccg tgtatgcgtt cgcgcgtgac 240

ggttttgcga ccaccgagga atttctgctg cgtctgggtc gtcacttcac cgaaggcttt 300

gactgggtta ccggtggccg ttgggcggcg cagcaattct tttgggatcg tatcaacgac 360

cacgatcacg cgttcagccg taacaaaagc gaggtgcgca ccgcggttct ggagattagc 420

ggcagcgaac aggcgatcgt ggcgggtatt gagggcctga ccgttctgaa gagcaccggt 480

agcgaattcc acggctttcc gcgtgacaaa tacaccaccc tgcaagagac caccgaccgt 540

attctggcga ccgatgtgag cgcgcgttgg cgttacaaca ccgtggaagt tgacttcgat 600

gcggtgtatg cgagcgttcg tggtctgctg ctgaaagcgt ttgcggagac ccacagcctg 660

gcgctgcagc aaaccatgta tgagatgggc cgtgcggtta ttgaaaccca cccggagatc 720

gatgaaatta agatgagcct gccgaacaaa caccacttcc tggtggacct gcagccgttt 780

ggtcaagata acccgaacga agttttctac gcggcggacc gtccgtatgg tctgatcgag 840

gcgaccattc aacgtgaagg cagccgtgcg gatcacccga tctggagcaa cattgcgggc 900

ttttgctaa 909

<210> 13

<211> 909

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

atgaccgcga ccgcggaaac cagcaccggt accaaggtgg ttctgggtca gaaccaatac 60

ggcaaggcgg aagtgcgtct ggtgaaagtt acccgtaaca ccgcgcgtca cgaaatccag 120

gacctgaacg ttaccagcca actgcgtggt gattttgagg cggcgcatac cgcgggtgac 180

aacgcgcatg tggttgcgac cgatacccag aagaacaccg tgtatgcgtt cgcgcgtgac 240

ggttttgcga ccaccgagga atttctgctg cgtctgggta aacacttcac cgaaggcttt 300

gactgggtta ccggtggccg ttgggcggcg cagcaattct tttgggatcg tatcaacgac 360

cacgatcacg cgttcagccg taacaagagc gaggtgcgca ccgcggttct ggagattagc 420

ggcagcgaac aggcgatcgt ggcgggtatt gagggcctga ccgttctgaa aagcaccggt 480

agcgaattcc acggctttcc gcgtgaccgt tacaccaccc tgcaagagac caccgaccgt 540

attctggcga ccgatgtgag cgcgcgttgg cgttacaaca ccgtggaagt tgacttcgat 600

gcggtgtatg cgagcgttcg tggtctgctg ctgaaagcgt ttgcggagac ccacagcctg 660

gcgctgcagc aaaccatgta tgagatgggc cgtgcggtta ttgaaaccca cccggagatc 720

gatgaaatta agatgagcct gccgaacaaa caccacttcc tggtggacct gcagccgttt 780

ggtcaagata acccgaacga agttttctac gcggcggacc gtccgtatgg tctgatcgag 840

gcgaccattc aacgtgaagg cagccgtgcg gatcacccga tctggagcaa cattgcgggc 900

ttttgctaa 909

<210> 14

<211> 909

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 14

atgaccgcga ccgcggaaac cagcaccggt accaaggtgg ttctgggtca gaaccaatac 60

ggcaaggcgg aagtgcgtct ggtgaaagtt acccgtaaca ccgcgcgtca cgaaatccag 120

gacctgaacg ttaccagcca actgcgtggt gattttgagg cggcgcatac cgcgggtgac 180

aacgcgcatg tggttgcgac cgatacccag aagaacaccg tgtatgcgtt cgcgcgtgac 240

ggttttgcga ccaccgagga atttctgctg cgtctgggta aacacttcac cgaaggcttt 300

gactgggtta ccggtggccg ttgggcggcg cagcaattct tttgggatcg tatcaacgac 360

cacgatcacg cgttcagccg taacaaaagc gaggtgcgca ccgcggttct ggagattagc 420

ggcagcgaac aggcgatcgt ggcgggtatt gagggcctga ccgttctgaa gagcaccggt 480

agcgaattcc acggctttcc gcgtgacaaa tacaccaccc tgcaagagac caccgaccgt 540

attctggcga ccgatgtgag cgcgcgttgg cgttacaaca ccgtggaagt tgacttcgat 600

gcggtgtatg cgagcgttcg tggtctgctg ctgcgtgcgt ttgcggagac ccacagcctg 660

gcgctgcagc aaaccatgta tgagatgggc cgtgcggtta ttgaaaccca cccggagatc 720

gatgaaatta agatgagcct gccgaacaaa caccacttcc tggtggacct gcagccgttt 780

ggtcaagata acccgaacga agttttctac gcggcggacc gtccgtatgg tctgatcgag 840

gcgaccattc aacgtgaagg cagccgtgcg gatcacccga tctggagcaa cattgcgggc 900

ttttgctaa 909

<210> 15

<211> 909

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 15

atgaccgcga ccgcggaaac cagcaccggt accaaggtgg ttctgggtca gaaccaatac 60

ggcaaggcgg aagtgcgtct ggtgaaagtt acccgtaaca ccgcgcgtca cgaaatccag 120

gacctgaacg ttaccagcca actgcgtggt gattttgagg cggcgcatac cgcgggtgac 180

aacgcgcatg tggttgcgac cgatacccag aagaacaccg tgtatgcgtt cgcgcgtgac 240

ggttttgcga ccaccgagga atttctgctg cgtctgggta aacacttcac cgaaggcttt 300

gactgggtta ccggtggccg ttgggcggcg cagcaattct tttgggatcg tatcaacgac 360

cacgatcacg cgttcagccg taacaaaagc gaggtgcgca ccgcggttct ggagattagc 420

ggcagcgaac aggcgatcgt ggcgggtatt gagggcctga ccgttctgaa gagcaccggt 480

agcgaattcc acggctttcc gcgtgacaaa tacaccaccc tgcaagagac caccgaccgt 540

attctggcga ccgatgtgag cgcgcgttgg cgttacaaca ccgtggaagt tgacttcgat 600

gcggtgtatg cgagcgttcg tggtctgctg ctgaaggcgt ttgcggagac ccacagcctg 660

gcgctgcagc aaaccatgta tgagatgggc cgtgcggtta ttgaaaccca cccggagatc 720

gatgaaattc gtatgagcct gccgaacaaa caccacttcc tggtggacct gcagccgttt 780

ggtcaagata acccgaacga agttttctac gcggcggacc gtccgtatgg tctgatcgag 840

gcgaccattc aacgtgaagg cagccgtgcg gatcacccga tctggagcaa cattgcgggc 900

ttttgctaa 909

<210> 16

<211> 909

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

atgaccgcga ccgcggaaac cagcaccggt accaaggtgg ttctgggtca gaaccaatac 60

ggcaaggcgg aagtgcgtct ggtgaaagtt acccgtaaca ccgcgcgtca cgaaatccag 120

gacctgaacg ttaccagcca actgcgtggt gattttgagg cggcgcatac cgcgggtgac 180

aacgcgcatg tggttgcgac cgatacccag aagaacaccg tgtatgcgtt cgcgcgtgac 240

ggttttgcga ccaccgagga atttctgctg cgtctgggta aacacttcac cgaaggcttt 300

gactgggtta ccggtggccg ttgggcggcg cagcaattct tttgggatcg tatcaacgac 360

cacgatcacg cgttcagccg taacaaaagc gaggtgcgca ccgcggttct ggagattagc 420

ggcagcgaac aggcgatcgt ggcgggtatt gagggcctga ccgttctgaa gagcaccggt 480

agcgaattcc acggctttcc gcgtgacaaa tacaccaccc tgcaagagac caccgaccgt 540

attctggcga ccgatgtgag cgcgcgttgg cgttacaaca ccgtggaagt tgacttcgat 600

gcggtgtatg cgagcgttcg tggtctgctg ctgaaggcgt ttgcggagac ccacagcctg 660

gcgctgcagc aaaccatgta tgagatgggc cgtgcggtta ttgaaaccca cccggagatc 720

gatgaaatta aaatgagcct gccgaaccgt caccacttcc tggtggacct gcagccgttt 780

ggtcaagata acccgaacga agttttctac gcggcggacc gtccgtatgg tctgatcgag 840

gcgaccattc aacgtgaagg cagccgtgcg gatcacccga tctggagcaa cattgcgggc 900

ttttgctaa 909

Claims

1. A method for purifying uricase, characterized in that the method comprises the steps of: purifying the sample containing uricase to be purified by hydrophobic chromatography and anion exchange chromatography respectively;

The hydrophobic chromatography comprises the steps of first balancing, loading, second balancing, eluting impurities and protein leaching; the mobile phase used in the first balance, the second balance, the eluting impurity and the protein eluting step contains salt, wherein the salt concentration in the mobile phase used in the second balance, the eluting impurity and the protein eluting step is sequentially reduced;

The salt used in the mobile phase in the hydrophobic chromatography is selected from one or more of (NH ₄)₂SO₄、Na₂SO₄、K₂SO₄、NH₄ Cl, naCl, and KCl;

The concentration of mobile phase salt used in the protein dissolution step in the hydrophobic chromatography is 0.01-3M;

The amino acid sequence of the uricase is shown as SEQ ID NO.4 or 8.

2. The purification method of claim 1, further comprising a step of sample treatment prior to purification, the step of sample treatment comprising mixing a sample treatment solution with the sample to be purified and collecting crude uricase for the purification step;

The anions of the salt of the sample treatment solution are selected from one or more of PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-、SO₄ ^2-、CH₃COO^-、F^-、Cl^-、Br^-、NO₃ ^-、ClO₄ ^-、I^- and SCN ^-; the cations of the salts of the sample processing solutions include one or more of NH₄ ⁺、Rb⁺、K⁺、Na⁺、Cs⁺、Li⁺、Mg²⁺、Ca²⁺ and Ba ²⁺.

3. The purification method of claim 2, wherein the salt of the sample processing solution is selected from one or more of (NH ₄)₂SO₄、Na₂SO₄、K₂SO₄、NH₄ Cl, naCl, and KCl.

4. The purification method of claim 2, wherein the salt of the sample processing solution is selected from one or more of (NH ₄)₂SO₄, naCl, and KCl).

5. The purification method of claim 2, wherein the salt of the sample processing solution is (NH ₄)₂SO₄.

6. The purification method according to claim 2, wherein the salt concentration in the mixture after the sample treatment solution is mixed with the sample to be purified is 0.05 to 3M.

7. The purification method according to claim 2, wherein the salt concentration in the mixture after the sample treatment solution is mixed with the sample to be purified is 0.2 to 2M.

8. The purification method according to claim 2, wherein the salt concentration in the mixture after the sample treatment solution is mixed with the sample to be purified is 0.5 to 1.5M.

9. The purification method according to claim 2, wherein the salt concentration in the mixture after the sample treatment solution is mixed with the sample to be purified is 0.75 to 1.25M.

10. The purification method according to claim 1, wherein the salt used for the mobile phase in the hydrophobic chromatography is selected from one or more of (NH ₄)₂SO₄, naCl, and KCl.

11. The purification method according to claim 1, wherein the salt used in the mobile phase in the hydrophobic chromatography is (NH ₄)₂SO₄.

12. The purification method according to claim 1, wherein the mobile phase salt concentration used in the protein elution step in the hydrophobic chromatography is 0.05 to 2M.

13. The purification method according to claim 1, wherein the mobile phase salt concentration used in the protein elution step in the hydrophobic chromatography is 0.1 to 1M.

14. The purification method according to claim 1, wherein the mobile phase salt concentration used in the protein elution step in the hydrophobic chromatography is 0.2 to 0.8M.

15. The purification method of claim 1, wherein the anion exchange chromatography comprises the steps of first equilibration, loading, second equilibration, eluting impurities, and protein digestion; the mobile phase used in the eluting impurity and protein eluting step contains salt, wherein the salt concentration in the mobile phase used in the protein eluting step is higher than in the eluting impurity step.

16. The purification method according to claim 15, wherein the salt of the mobile phase used in anion exchange chromatography is selected from one or more of (NH ₄)₂SO₄、Na₂SO₄、K₂SO₄、NH₄ Cl, naCl, and KCl.

17. The purification method according to claim 15, wherein the salt of the mobile phase used in anion exchange chromatography is selected from one or more of (NH ₄)₂SO₄, naCl, and KCl).

18. The purification method according to claim 15, wherein the salt of the mobile phase used in anion exchange chromatography is NaCl.

19. The purification method according to claim 15, wherein the mobile phase salt concentration used in the protein elution step in the anion exchange chromatography is 0.01 to 2M.

20. The purification method according to claim 15, wherein the mobile phase salt concentration used in the protein elution step in the anion exchange chromatography is 0.05 to 1M.

21. The purification method according to claim 15, wherein the mobile phase salt concentration used in the protein elution step in the anion exchange chromatography is 0.1 to 0.5M.

22. The purification method according to claim 15, wherein the mobile phase salt concentration used in the protein elution step in the anion exchange chromatography is 0.2 to 0.3M.

23. A protein, characterized in that the amino acid sequence of the protein is as shown in SEQ ID NO:4 or 8.

24. A method for preparing a uricase conjugate, comprising the step of conjugating uricase to a polymer, wherein uricase is prepared by the purification method of any one of claims 1-22.

25. The method of claim 24, wherein the polymer is polyethylene glycol.

26. A process for the preparation of a composition comprising a conjugate of uricase, comprising the step of conjugating uricase to a polymer and mixing with pharmaceutically acceptable excipients, wherein uricase is prepared by the purification process of any one of claims 1-22.

27. The method of claim 26, wherein the polymer is polyethylene glycol.