A method for purifying uricase

Abstract

The present disclosure relates to a method for preparing a uricase variant and a novel method for purifying a uricase. Specifically, the uricase variant has higher activity and stability than the wild-type uricase; the purification method reconstructs the purification steps compared to the traditional method, and brings about a better purification effect through ammonium sulfate fractionation precipitation, dialysis, chromatography, secondary dialysis, ultrafiltration concentration and freeze drying.

Description

Technical Field

[0001] The present invention relates to a uricase variant and a method for purifying uricase. Background Art

[0002] Uric acid is the ultimate product of purine metabolism in organisms and can be degraded into allantoin, carbon dioxide and hydrogen peroxide by uricase. Uric acid is a poorly soluble substance. Only a small amount of uric acid is excreted from the body with urine and feces, and most of the uric acid is still stored in the human body. The rate of uric acid production and excretion in a normal human body is basically constant. With the improvement of living standards, people's lifestyles have changed greatly. The content of purine intake by the human body has continued to increase, resulting in an increase in the content of uric acid produced. However, the amount of uric acid excreted by the human body cannot increase accordingly, so a large amount of uric acid accumulates in the human body, resulting in an increasing incidence of diseases such as gout. Uricase can degrade uric acid and can effectively reduce the concentration of uric acid in the human body. However, the half-life of uricase in the human body is relatively short, and patients need to inject exogenous uricase for a long time and regularly, so the demand for uric acid continues to increase. Selecting the use of microbial fermentation production is the best method to obtain a large amount of uricase, but there are many impurities in the uricase obtained by this method, which needs to be purified before it can be used for the treatment of diseases.

[0003] At present, there are many ways to purify uricase in the prior art. CN201410015218.4 discloses a method for preparing natural mammalian uricase, which first homogenizes and crushes the mammalian liver, then washes it with a buffer solution of pH 8.2, and dissolves the washed precipitate with a buffer solution of pH 10.2 to obtain a preliminary extract of uricase; the preliminary extract is graded precipitated with ammonium sulfate, dissolved in a re-dissolving buffer solution, and the re-dissolving solution is purified by anion exchange chromatography (the anion exchange chromatography purification medium is an agarose series ion exchange medium coupled with a diethyl (2-hydroxypropyl) aminoethyl or diethylaminoethyl group) to obtain a crude pure solution of uricase; the above crude pure solution of uricase is purified by affinity chromatography to obtain a pure solution of uricase. This method is used for the purification of mammalian uricase, and only ammonium sulfate graded precipitation and affinity chromatography are performed, resulting in a large loss of enzyme activity; no treatment is performed after affinity chromatography, and energy consumption is required for freeze-drying, which takes a long time.

[0004] There are also multiple purification methods, such as polyethyleneimine, ammonium sulfate salting out, anion exchange chromatography, cation exchange chromatography, chromatography desalting, and freeze drying. However, the purification process completely ignores the effect of residual salt in the enzyme solution on anion exchange chromatography and cation exchange chromatography, which will lead to a worse effect of the purification process. In addition, anion exchange chromatography and cation exchange chromatography were performed at the same time, which did not improve the purification effect much, but increased the loss of uricase.

[0005] Currently, a variety of human uricases have appeared. For example, CN201510555258.2 discloses a recombinant human / baboon chimeric uricase protein, which treats gout by combining human and baboon chimeric uricases. However, there is still room for improvement in its activity. Summary of the invention

[0006] In order to solve the above problems, the present invention proposes a uricase variant and a new purification method. The uricase variant disclosed in the present invention has higher enzyme activity and better stability than wild-type uricase; the uricase purification method disclosed in the present invention can achieve purification with higher efficiency and better effect.

[0007] In one aspect of the present invention, a uricase variant is provided, wherein the variant has an amino acid sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to the wild-type uricase of SEQ ID NO.1.

[0008] In some specific embodiments, the uricase variant has at least one or more mutations at the following positions relative to the wild-type uricase with the amino acid sequence of SEQ ID NO.1: L181 and / or P182.

[0009] In some embodiments, the variant has at least one or more mutations at the following positions relative to the wild-type uricase variant with an amino acid sequence of SEQ ID NO.1: L181S and / or P182K.

[0010] In one aspect of the present invention, a nucleotide is provided, which encodes the uricase variant as described above.

[0011] In some embodiments, the nucleotides are codon optimized for expression in prokaryotes.

[0012] In some embodiments, the nucleotides are codon optimized for expression in E. coli.

[0013] In one aspect of the present invention, a vector is provided, wherein the vector comprises the aforementioned nucleotide.

[0014] In some embodiments, the nucleotide is operably linked to a promoter.

[0015] In some embodiments, the promoter is a constitutive promoter, an inducible promoter, a ubiquitous promoter, a cell type specific promoter, or a tissue specific promoter.

[0016] In one aspect of the present invention, a cell is provided, comprising the aforementioned uricase variant, nucleotide or vector.

[0017] In some embodiments, the cell is a prokaryotic cell.

[0018] In some embodiments, the cell is Escherichia coli.

[0019] In one aspect of the present invention, a method for purifying a uricase variant is provided, the steps of which are as follows:

[0020] (1) Ammonium sulfate graded precipitation

[0021] The fermentation liquid containing uricase after simple filtration is placed in ice water, and ammonium sulfate is slowly added, and the mixture is continuously stirred until the concentration of ammonium sulfate reaches 20%-40%; the mixture is ice-water bathed for 3 hours, and the mixture is centrifuged at 4°C and 12000 r / min for 15 minutes, and the supernatant is retained; then ammonium sulfate is slowly added to the supernatant, and the mixture is continuously stirred until the concentration of ammonium sulfate reaches 70%-90%; the mixture is ice-water bathed for 24 hours, and the mixture is centrifuged at 4°C and 12000 r / min for 15 minutes, and the precipitate is retained; the precipitate is then dissolved with 1-100 mM, pH 5.0-8.0 PBS buffer, and the mixture is stored in a refrigerator at 2-8°C;

[0022] (2) Dialysis

[0023] The enzyme solution obtained in step (1) is placed in a dialysis bag, which is sealed. The dialysis bag is then placed in a 1-100 mM, pH 5.0-8.0 PBS buffer solution and dialyzed for 24 h, with the dialysate being changed every 4 h.

[0024] (3) DEAE cellulose chromatography

[0025] After the DEAE cellulose chromatography column is equilibrated with a 1-100 mM PBS buffer at a pH of 5.0-8.0, the enzyme solution obtained in step (2) is loaded, and the column is equilibrated with the PBS buffer again, and then eluted with a 1-100 mM PBS buffer containing 0.5 M NaCl and a pH of 5.0-8.0, and the eluted sample is collected;

[0026] (4) Second dialysis

[0027] The enzyme solution obtained in step (3) is placed in a dialysis bag, which is sealed, and then the dialysis bag is placed in a 1-100 mM, pH 5.0-8.0 PBS buffer solution, and dialyzed for 24 hours, with the dialysate being changed every 4 hours;

[0028] (5) Ultrafiltration concentration

[0029] Performing membrane filtration on the enzyme solution obtained in step (4);

[0030] (6) Freeze-drying

[0031] The enzyme solution obtained in step (5) is placed in a -80°C refrigerator and frozen overnight, and then freeze-dried to obtain uricase enzyme powder.

[0032] In some specific embodiments, the purification method comprises the following steps:

[0033] (1) Ammonium sulfate graded precipitation

[0034] Place 100 mL of fermentation liquid containing uricase after simple filtration in ice water, slowly add 16.4 g of ammonium sulfate, and stir continuously until the ammonium sulfate is completely dissolved, so that the concentration of ammonium sulfate reaches 30%; ice water bath for 3 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the supernatant; then slowly add 34.8 g of ammonium sulfate to the supernatant, and stir continuously until the ammonium sulfate is completely dissolved, so that the concentration of ammonium sulfate reaches 80%; ice water bath for 24 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the precipitate; then dissolve the precipitate with 20 mL of 10 mM, pH 8.0 PBS buffer, and store in a refrigerator at 2-8°C;

[0035] (2) Dialysis

[0036] The enzyme solution obtained in step (1) was placed in a dialysis bag, which was sealed. The dialysis bag was then placed in a 10 mM, pH 8.0 PBS buffer solution and dialyzed for 24 h, with the dialysate being changed every 4 h.

[0037] (3) DEAE cellulose chromatography

[0038] After the DEAE cellulose chromatography column is equilibrated with 10 mM PBS buffer at pH 8.0, the enzyme solution obtained in step (2) is loaded, and then equilibrated with the PBS buffer, and then eluted with 10 mM PBS buffer containing 0.5 M NaCl and pH 8.0, and the eluted sample is collected;

[0039] (4) Second dialysis

[0040] The enzyme solution obtained in step (3) was placed in a dialysis bag, which was sealed. The dialysis bag was then placed in a 10 mM, pH 8.0 PBS buffer and dialyzed for 24 h, with the dialysate being changed every 4 h.

[0041] (5) Ultrafiltration concentration

[0042] Performing membrane filtration on the enzyme solution obtained in step (4);

[0043] (6) Freeze-drying

[0044] The enzyme solution obtained in step 5 is placed in a -80°C refrigerator and frozen overnight, and then freeze-dried to obtain uricase enzyme powder.

[0045] In some embodiments, the purification method comprises the following steps:

[0046] (1) Ammonium sulfate graded precipitation

[0047] Place 100 mL of fermentation liquid containing uricase after simple filtration in ice water, slowly add 22.6 g of ammonium sulfate, and stir continuously until the ammonium sulfate is completely dissolved, so that the concentration of ammonium sulfate reaches 20%. Ice water bath for 3 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the supernatant. Then slowly add 25.0 g of ammonium sulfate to the supernatant, and stir continuously until the ammonium sulfate is completely dissolved, so that the concentration of ammonium sulfate reaches 70%; ice water bath for 24 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the precipitate; then dissolve the precipitate with 20 mL of 100 mM, pH 7.0 PBS buffer, and store in a refrigerator at 2-8°C;

[0048] (2) Dialysis

[0049] The enzyme solution obtained in step (1) was placed in a dialysis bag, which was sealed. The dialysis bag was then placed in a 100 mM, pH 7.0 PBS buffer solution and dialyzed for 24 h, with the dialysate being changed every 4 h.

[0050] (3) DEAE cellulose chromatography

[0051] After the DEAE cellulose chromatography column is equilibrated with 100 mM PBS buffer at pH 7.0, the enzyme solution obtained in step (2) is loaded, and then equilibrated with the PBS buffer, and then eluted with 100 mM PBS buffer containing 0.5 M NaCl and pH 7.0, and the eluted sample is collected;

[0052] (4) Second dialysis

[0053] The enzyme solution obtained in step (3) was placed in a dialysis bag, which was sealed. The dialysis bag was then placed in a 100 mM, pH 7.0 PBS buffer solution and dialyzed for 24 h, with the dialysate being changed every 4 h.

[0054] (5) Ultrafiltration concentration

[0055] The enzyme solution obtained in step 4 is subjected to membrane filtration;

[0056] (6) Freeze-drying

[0057] The enzyme solution obtained in step 5 is placed in a -80°C refrigerator and frozen overnight, and then freeze-dried to obtain uricase enzyme powder.

[0058] In some embodiments, the purification method comprises the following steps:

[0059] (1) Ammonium sulfate graded precipitation

[0060] Place 100 mL of fermentation liquid containing uricase after simple filtration in ice water, slowly add 10.6 g of ammonium sulfate, and stir continuously until the ammonium sulfate is completely dissolved, so that the concentration of ammonium sulfate reaches 20%. Place in ice water bath for 3 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the supernatant; then slowly add 49.7 g of ammonium sulfate to the supernatant, and stir continuously until the ammonium sulfate is completely dissolved, so that the concentration of ammonium sulfate reaches 90%; place in ice water bath for 24 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the precipitate; then dissolve the precipitate with 20 mL of 1 mM, pH 6.0 PBS buffer, and store in a refrigerator at 2-8°C;

[0061] (2) Dialysis

[0062] The enzyme solution obtained in step (1) was placed in a dialysis bag, which was sealed. The dialysis bag was then placed in a 1 mM, pH 6.0 PBS buffer and dialyzed for 24 h, with the dialysate being changed every 4 h.

[0063] (3) DEAE cellulose chromatography

[0064] After the DEAE cellulose chromatography column is equilibrated with 100 mM PBS buffer at pH 7.0, the enzyme solution obtained in step (2) is loaded, and then equilibrated with the PBS buffer, and then eluted with 1 mM PBS buffer containing 0.5 M NaCl and pH 6.0, and the eluted sample is collected;

[0065] (4) Second dialysis

[0066] The enzyme solution obtained in step (3) was placed in a dialysis bag, which was sealed. The dialysis bag was then placed in a 1 mM, pH 6.0 PBS buffer and dialyzed for 24 h, with the dialysate being changed every 4 h.

[0067] (5) Ultrafiltration concentration

[0068] Performing membrane filtration on the enzyme solution obtained in step 4;

[0069] (6) Freeze-drying

[0070] The enzyme solution obtained in step 5 is placed in a -80°C refrigerator and frozen overnight, and then freeze-dried to obtain uricase enzyme powder.

[0071] In one aspect of the present invention, provided is the use of the aforementioned uricase variant in the preparation of a medicament.

[0072] In some embodiments, the medicament is used to treat gout and / or reduce uric acid levels.

[0073] In one aspect of the present invention, a non-therapeutic method for reducing uric acid levels in mammalian body fluids or tissues is provided, comprising the steps of: administering the aforementioned uricase variant to a subject; and detecting the uric acid level in the body fluids or tissues of the subject.

[0074] In some embodiments, the mammal comprises a human.

[0075] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0076] (1) The present invention discovers a uricase variant through mutation, which has higher enzyme activity (titer) and better stability than the wild-type uricase.

[0077] (2) The purification method adopted by the present invention is simple and efficient, and has better purification effect than the prior art, and can purify uricase efficiently.

[0078] (3) The uricase prepared by the present invention can achieve better therapeutic effects and has potential application prospects. DETAILED DESCRIPTION

[0079] The following examples are only used to describe the present invention, but not to limit the present invention. Unless otherwise specified, the experiment and method described in the embodiment are carried out basically according to the conventional methods well known in the art and described in various references. For example, the conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, "Molecular cloning: laboratory manual" (MOLECULAR CLONING: A LABORATORY MANUAL), 2nd edition (1989); "Contemporary molecular biology experimental manual" (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (FM Ausubel et al. edited, (1987)); "Enzymology" (METHODS IN ENZYMOLOGY) series (Academic Publishing Company): "PCR 2: Practical methods" (PCR 2: APRACTICAL APPROACH) (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), ANTIBODIES, A LABORATORY MANUAL, eds. Harlow and Lane, eds. (1988), and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0080] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0081] Uricase variants

[0082] The uricase variant provided by the present invention has higher enzyme activity and better stability than wild-type uricase.

[0083] Uricase purification

[0084] The uricase purification method disclosed in the present invention can achieve purification with higher efficiency and better effect.

[0085] Definition of terms

[0086] Unless otherwise specified, all technical and scientific terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. For the purpose of the present invention, the following terms are defined to be consistent with the meanings commonly understood in the art.

[0087] In the present invention, the articles "a / an" and "the" are used to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "element" means one element or more than one element. The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives.

[0088] In the present invention, the term "and / or" should be understood to mean either or both of the alternatives.

[0089] In the present invention, the term "amino acid" means twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0090] In some embodiments, the selected group of amino acids that are considered to be conservative substitutions for each other

[0091] Acidic residue D and E Basic residues K, R and H Hydrophilic uncharged residues S, T, N and Q Aliphatic uncharged residue G, A, V, L, and I Nonpolar uncharged residues C, M and P Aromatic residues F, Y and W

[0092] In some embodiments, a selected group of amino acids that are considered to be conservative substitutions for each other:

[0093] Group 1 A, S and T Group 2 D and E Group 3 N and Q Group 4 R and K Group 5 1. L and M Group 6 F, Y and W

[0094] In certain embodiments, other selected groups of amino acids that are considered conservative substitutions for each other are:

[0095]

[0096]

[0097] In the present invention, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. It is a replicon, such as a plasmid, phage or cosmid, into which another DNA segment can be inserted to achieve replication of the inserted segment. Generally, a vector is capable of replication when combined with appropriate control elements.

[0098] In some cases, the vector system comprises a single vector. Alternatively, the vector system comprises multiple vectors. The vector can be a viral vector. Vectors include, but are not limited to, single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA or both; and other polynucleotide variants known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, into which other DNA segments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a DNA or RNA sequence of viral origin is present in the vector for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). Viral vectors also include polynucleotides carried by viruses for transfection into host cells.

[0099] Some vectors can replicate autonomously (for example, bacterial vectors and free-type mammalian vectors with bacterial replication origin) in the host cell in which they are introduced. After being introduced into the host cell, other vectors (for example, non-free-type mammalian vectors) are integrated into the genome of the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of the gene operably connected thereto. Such vectors are referred to as "expression vectors" in the present invention. The vectors expressed in eukaryotic cells and the vectors causing expression in eukaryotic cells may be referred to as "eukaryotic expression vectors" in this article. Common expression vectors useful in recombinant DNA technology are generally in the form of plasmids. Recombinant expression vectors can be suitable for comprising nucleic acids of the present invention in the form of nucleic acids expressed in host cells, which means that recombinant expression vectors include one or more regulatory elements, which can be selected according to the host cell to be used for expression, and the nucleic acid is operably connected to the nucleic acid sequence to be expressed. In recombinant expression vectors, "operably connected" is intended to refer to that the target nucleotide sequence is connected to the regulatory element in a manner that allows the expression of the nucleotide sequence (for example, in an in vitro transcription / translation system or in a host cell when the vector is introduced into a host cell). Advantageous vectors include lentiviruses and adeno-associated viruses, and the type of these vectors can also be selected to target specific types of cells.

[0100] In the present invention, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES) and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements include those that direct constitutive expression of nucleotide sequences in many types of host cells and those that direct expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in target desired tissues such as muscle, neurons, bones, skin, blood, specific organs (e.g., liver, pancreas) or specific cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific.

[0101] In the present invention, the term "uric acid" refers to a heterocyclic compound containing carbon, nitrogen, oxygen and hydrogen, and its molecular formula is C5H4N4O3. Uric acid is the final metabolite of purine in the human body.

[0102] The term "Hyperuricemia (HUA)" refers to the condition in which the fasting blood uric acid level is higher than 420 μmol / L (7 mg / dL) for men and higher than 360 μmol / L (6 mg / dL) for women on two different days under a normal purine diet. Hyperuricemia is the basis for gout, but it is not enough to cause gout. Gout only occurs when urate is deposited in body tissues and causes damage.

[0103] The term "asymptomatic hyperuricemia" refers to patients who only have hyperuricemia but no clinical symptoms such as arthritis, tophi, and uric acid stones.

[0104] The term "gout" is a crystal-related joint disease caused by urate deposition, which is directly related to hyperuricemia caused by purine metabolism disorders and (or) reduced uric acid excretion, and belongs to the category of metabolic rheumatic diseases. Gout can be complicated by kidney disease, and in severe cases, joint destruction and renal damage may occur. It is often accompanied by hyperlipidemia, hypertension, diabetes, arteriosclerosis and coronary heart disease. Gout patients often experience sudden joint pain at night, with severe pain, edema, redness, swelling and inflammation in the joints. Joint pain often occurs in the first metatarsal bone, and can also occur in the hand joints, knees, elbows, etc. Subsequently, the pain gradually subsides until it disappears, lasting for a few days or weeks.

[0105] The term "uricase" refers to an enzyme that degrades uric acid.

[0106] The term "animal model" refers to animals that are used in various medical and scientific research to mimic human disease.

[0107] Example

[0108] Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0109] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0110] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0111] Example 1 Obtaining uricase variants

[0112] For the known uricase protein (the recombinant human / baboon chimeric uricase protein in CN201510555258.2, referred to as wild-type uricase in this embodiment), the key amino acid sites that may affect its biological function are predicted by bioinformatics, and the amino acid sites are mutated to obtain uricase mutants. The amino acid sequence of wild-type uricase is shown in SEQ ID No.1, and its nucleotide sequence is shown in SEQ ID No.2. The amino acid sites potentially related to the function of uricase are site-directed mutated by bioinformatics methods.

[0113] SEQ ID NO.1:

[0114] MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIEAFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQMKSGPPV IHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLKKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL

[0115] SEQ ID NO.2:

[0116] ATGGCCCATTATCACAACGACTATAAGAAAAATGATGAAGTGGAGTTTGTGCGCACCGGCTATGGCAAGGACATGGTGAAAGTGCTGCACATTCAGCGCGATGGTAAGTACCACAGCATCAAAGAAGTGGCCACCAGTGTGCAGCTGACCCTGAGCAGCAAAAAGGATTATCTGCACGGCGACAACAGCGATATCATTCCGACCGATACCATTAAAAATACCGTGCATGTTCTGGCCAAATTCAAGGGCATTAAGAGCATCGAGGCCTTTGCCATGAACATCTGCGAACACTTCCTGAGCAGCTTCAACCACGTGATCCGTGCCCAGGTGTATGTGGAGGAAGTGCCGTGGAAACGCTTCGAGAAAAACGGCGTGAAGCATGTTCACGCCTTCATTCACACCCCTACCGGCACCCACTTCTGCGAGGTGGAACAGATGAAAAGCGGCCCGCCGGTTATTCACAGCGGTATCAAAGATTTAAAAGTTCTGAAGACAACCCAGAGCGGCTTTGAAGGCTTCATCAAGGACCAGTTTACCACCCTGCCGGAAGTTAAGGACCGCTGTTTCGCCACCCAGGTTTACTGCAAGTGGCGTTATCACCAGGGTCGCGATGTTGACTTTGAAGCCACCTGGGATACCGTGCGCGACATCGTGCTGAAAAAGTTCGCCGGTCCGTATGACAAGGGTGAGTATAGCCCGAGTGTGCAGAAAACCCTGTACGATATTCAGGTGCTGAGCCTGAGCCGCGTGCCGGAGATTGAAGACATGGAGATCAGCCTGCCGAACATCCATTATTTTAACATCGATATGAGCAAAATGGGCCTGATCAACAAGGAAGAGGTGCTGCTGCCGTTAGATAACCCGTATGGCAAGATCACCGGCACCGTTAAACGCAAACTGAGTAGCCGCCTG

[0117] Amino acid site-directed mutagenesis was performed by PCR, specifically, primers were set at the position of the mutated amino acid codon of the wild-type uricase nucleotide, the codon was changed to the mutated amino acid codon, and then the various PCR fragments were homologously recombined by Gibsonclone to obtain a mutant nucleotide sequence with the mutated amino acid. The specific mutation information of the amino acid is as follows:

[0118]

[0119] Through the above method, three mutants were obtained, including L181S, P182K and L181S+P182K.

[0120] Example 2 Mutant Activity Verification

[0121] 1. Preparation of wild-type uricase and mutant uricase cloning plasmids and expression bacteria

[0122] The nucleotide sequences encoding the L181S mutant (SEQ ID NO.3), the P182K mutant (SEQ ID NO.4) and the L181S+P182K mutant (SEQ ID NO.5) were chemically synthesized, and the nucleotide sequences were connected with the linearized pET28a plasmid (purchased from Nanjing Novozyme) to obtain the recombinant plasmid, and the recombinant plasmid was transformed into Escherichia coli DH5α (purchased from Nanjing Novozyme). The pET28a plasmid was extracted from the positive transformant, and the pET28a plasmid was transformed into Escherichia coli BL21 by heat shock transformation. The transformation product was transferred to LB medium to obtain the transformant Escherichia coli. The details are as follows:

[0123] A gene encoding a fusion protein uricase whose nucleotide sequence is shown in SEQ ID NO.2 is synthesized; PCR amplification is performed using an upstream primer and a downstream primer with the synthesized gene as a template, and the amplified product is recovered by agarose gel electrophoresis; the amplified product and the pET28a plasmid are respectively digested with restriction endonucleases NdeⅠ / XhoⅠ (purchased from NEB Company), and the digested products are recovered by gel and connected to obtain a connection product; the connection product is transformed into Escherichia coli DH5α (purchased from Beijing Qingke Biotechnology Co., Ltd.) to obtain a transformation product.

[0124] 2. Spread the transformation product on LB solid medium containing 100μg / mL kanamycin, invert and culture in a 37℃ constant temperature incubator for 12h to obtain transformants; pick the transformants and inoculate them into 5mL LB liquid medium containing 100μg / mL kanamycin, shake the flask at 37℃ and 180rpm for 12h, extract the plasmid for enzyme verification and sequence and compare with the target gene, and obtain the recombinant plasmid if the verification is correct. Take 50μL of E. coli BL21 competent, ice bath, add 2μL of recombinant plasmid, ice bath for 15min, heat shock at 42℃ for 90s, ice bath for 3min, add 600μL of LB, shake at 37℃ shaker at 220rpm for 1h. Take 100μL and evenly spread it on the LB plate containing kanamycin resistance, incubate at 37℃, first incubate upright for 10min, then invert the solid plate in a 37℃ incubator overnight to obtain transformant E. coli. Identification of transformant E. coli: The kanamycin resistance gene on the pET28a plasmid was used as a marker for transformant identification.

[0125] Example 3 Expression of wild-type uricase and mutant uricase

[0126] 1. Pick the verified wild type and mutant transformant single colonies and inoculate them into 50mL LB liquid culture medium containing 100μg / mL kanamycin, and shake culture at 37℃ and 180rpm for 12h to obtain culture solution; transfer the culture solution to 1000mL LB liquid culture medium containing 100μg / mL kanamycin at a ratio of 1:50, shake culture at 37℃ and 220rpm for 4h until OD600=0.8, then reduce the temperature to 16℃, continue shaking culture at 180rpm for 1h, add IPTG (final concentration 1mM) after 1h, and culture at 16℃ and 180rpm for 12h to obtain expression bacteria.

[0127] 2. Purification of wild-type and mutant uricase

[0128] The correct wild type and mutant transformant single colonies were picked and inoculated into 50 mL of LB liquid culture medium containing 100 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 12 h to obtain culture solution; the culture solution was transferred to 1000 mL of LB liquid culture medium containing 100 μg / mL kanamycin at a ratio of 1:50, and cultured at 37°C and 220 rpm for 4 h until the OD600 value was 0.8, then the temperature was lowered to 16°C, and the culture was continued at 180 rpm for 1 h. After 1 h, isopropylthiogalactoside IPTG (final concentration 1 mM) was added, and cultured at 16°C and 180 rpm for 12 h to obtain expression bacteria. After collecting the wet cells, PBS buffer was added at a ratio of 1 g wet cells: 10 mL PBS buffer (pH 8.0, 50 mM) to suspend the cells. The bacterial suspension was ultrasonically disrupted in a 4°C low-temperature ice water bath. The ultrasonic disruption conditions were: power 250W, 3s on, 4s off, temperature 4°C, and effective disruption time 10min. The ultrasonically disrupted mixed solution was centrifuged at 15000rpm for 25min, and the supernatant was aspirated to obtain the crude enzyme solution. Different uricases were diluted to the same concentration (about 2.5mg / ml) using PBS.

[0129] 3. Detect the therapeutic effects of different uricases

[0130] Thirty-five healthy male Wistar rats (purchased from Jicui Yaokang), 10 weeks old and weighing 150±10g, were randomly divided into 7 groups, 5 rats in each group, including blank control group, model group, wild-type uricase group, L180S group, P182K group, L181S+P182K group, and benzbromarone group.

[0131] Potassium oxonate was selected as a uricase inhibitor and injected intraperitoneally to inhibit uricase in liver peroxisomes, increase blood uric acid and form hyperuricemia. Except for the blank control group, mice in other groups were given 25g / L potassium oxonate 10ml / kg, and gavage was given 1h later. The control group and model group were given sodium carboxymethyl cellulose emulsion (prepared with distilled water) 10ml / kg each time, twice a day; the wild-type uricase group, L180S group, P182K group, L181S+P182K group were given wild-type uricase, L180S uricase, P182K uricase, L181S+P182K uricase 10mg / kg, twice a day; the benzbromarone group was given benzbromarone emulsion suspension 5mL / kg, twice a day. One hour after intragastric administration, the rats were cut off from their tails to collect blood, and the blood samples were placed in 1.5 mL EP tubes, incubated at 37°C for 30 minutes, centrifuged to collect the supernatant, and the blood uric acid was measured using a biochemical analyzer using the uricase-EHSPT method. The test results are shown in the following table:

[0132]

[0133] It can be seen from the above table that oral administration of wild-type uricase and various mutant uricases can significantly reduce the blood uric acid content in rats. L180S, P182K, and L181S+P182K uricases can play a good role in lowering blood uric acid, especially L181S+P182K uricase, which can play the best role in lowering uric acid.

[0134] Example 4 Technical Effect Detection of Different Purification Methods

[0135] In order to detect the purification effect of different purification methods on uricase, the crude enzyme solution of L181S+P182K uricase obtained in step 2 of Example 2 was selected as the purification object. The crude enzyme solution was purified by the following purification methods:

[0136] 1. Method 1

[0137] (1) Ammonium sulfate graded precipitation: Place 100 mL of fermentation broth containing uricase after simple filtration in ice water, slowly add 16.4 g of ammonium sulfate, and stir continuously until the ammonium sulfate is completely dissolved and the concentration of ammonium sulfate reaches 30%. Place in an ice water bath for 3 hours, centrifuge at 4°C and 12,000 r / min for 15 minutes, and retain the supernatant. Then slowly add 34.8 g of ammonium sulfate to the supernatant and stir continuously until the ammonium sulfate is completely dissolved and the concentration of ammonium sulfate reaches 80%. Place in an ice water bath for 24 hours, centrifuge at 4°C and 12,000 r / min for 15 minutes, and retain the precipitate. Dissolve the precipitate with 20 mL of 10 mM, pH 8.0 PBS buffer and store in a refrigerator at 2-8°C.

[0138] (2) Dialysis: Place the enzyme solution obtained in step (1) in a dialysis bag, seal it, and then place the dialysis bag in 10 mM PBS buffer, pH 8.0. Dialyze for 24 h, changing the dialysis solution every 4 h.

[0139] (3) DEAE cellulose chromatography: After the DEAE cellulose chromatography column was equilibrated with 10 mM PBS buffer at pH 8.0, the enzyme solution obtained in step (2) was loaded, and the column was equilibrated with the PBS buffer again. The column was then eluted with 10 mM PBS buffer containing 0.5 M NaCl at pH 8.0, and the eluted sample was collected.

[0140] (4) Second dialysis: Place the enzyme solution obtained in step (3) in a dialysis bag, seal it, and then place the dialysis bag in 10 mM PBS buffer, pH 8.0. Dialyze for 24 hours, changing the dialysis solution every 4 hours.

[0141] (5) Ultrafiltration and concentration: The enzyme solution obtained in step 4 is subjected to membrane filtration.

[0142] (6) Freeze-drying: The enzyme solution obtained in step 5 was placed in a -80°C refrigerator and frozen overnight, and then freeze-dried to obtain 2.3 g of uricase enzyme powder with a potency of 6351.2 U / g.

[0143] 2. Method 2

[0144] (1) Ammonium sulfate graded precipitation: Place 100 mL of fermentation broth containing uricase after simple filtration in ice water, slowly add 22.6 g of ammonium sulfate, and stir continuously until the ammonium sulfate is completely dissolved and the concentration of ammonium sulfate reaches 20%. Place in an ice water bath for 3 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the supernatant. Then slowly add 25.0 g of ammonium sulfate to the supernatant and stir continuously until the ammonium sulfate is completely dissolved and the concentration of ammonium sulfate reaches 70%. Place in an ice water bath for 24 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the precipitate. Dissolve the precipitate with 20 mL of 100 mM, pH 7.0 PBS buffer and store in a refrigerator at 2-8°C.

[0145] (2) Dialysis: Place the enzyme solution obtained in step (1) in a dialysis bag, seal it, and then place the dialysis bag in 100 mM PBS buffer, pH 7.0. Dialyze for 24 h, changing the dialysis solution every 4 h.

[0146] (3) DEAE cellulose chromatography: After the DEAE cellulose chromatography column was equilibrated with 100 mM PBS buffer at pH 7.0, the enzyme solution obtained in step (2) was loaded, and the column was equilibrated with the PBS buffer again. The column was then eluted with 100 mM PBS buffer containing 0.5 M NaCl at pH 7.0, and the eluted sample was collected.

[0147] (4) Second dialysis: Place the enzyme solution obtained in step (3) in a dialysis bag, seal it, and then place the dialysis bag in 100 mM PBS buffer, pH 7.0. Dialyze for 24 h, changing the dialysis solution every 4 h.

[0148] (5) Ultrafiltration and concentration: The enzyme solution obtained in step 4 is subjected to membrane filtration.

[0149] (6) Freeze-drying: The enzyme solution obtained in step 5 was placed in a -80°C refrigerator and frozen overnight, and then freeze-dried to obtain 1.7 g of uricase powder with a potency of 7533.3 U / g.

[0150] 3. Method 3

[0151] (1) Ammonium sulfate graded precipitation: Place 100 mL of fermentation broth containing uricase after simple filtration in ice water, slowly add 10.6 g of ammonium sulfate, and stir continuously until the ammonium sulfate is completely dissolved and the concentration of ammonium sulfate reaches 20%. Place in an ice water bath for 3 hours, centrifuge at 4°C and 12,000 r / min for 15 minutes, and retain the supernatant. Then slowly add 49.7 g of ammonium sulfate to the supernatant and stir continuously until the ammonium sulfate is completely dissolved and the concentration of ammonium sulfate reaches 90%. Place in an ice water bath for 24 hours, centrifuge at 4°C and 12,000 r / min for 15 minutes, and retain the precipitate. Dissolve the precipitate with 20 mL of 1 mM, pH 6.0 PBS buffer and store in a refrigerator at 2-8°C.

[0152] (2) Dialysis: Place the enzyme solution obtained in step (1) in a dialysis bag, seal it, and then place the dialysis bag in 1 mM, pH 6.0 PBS buffer. Dialyze for 24 h, changing the dialysis solution every 4 h.

[0153] (3) DEAE cellulose chromatography: After the DEAE cellulose chromatography column was equilibrated with 100 mM PBS buffer at pH 7.0, the enzyme solution obtained in step (2) was loaded, and the column was equilibrated with the PBS buffer again. The column was then eluted with 1 mM PBS buffer containing 0.5 M NaCl at pH 6.0, and the eluted sample was collected.

[0154] (4) Second dialysis: Place the enzyme solution obtained in step (3) in a dialysis bag, seal it, and then place the dialysis bag in 1 mM, pH 6.0 PBS buffer. Dialyze for 24 h, changing the dialysis solution every 4 h.

[0155] (5) Ultrafiltration and concentration: The enzyme solution obtained in step 4 is subjected to membrane filtration.

[0156] (6) Freeze-drying: The enzyme solution obtained in step 5 was placed in a -80°C refrigerator and frozen overnight, and then freeze-dried to obtain 2.5 g of uricase enzyme powder with a potency of 5217.1 U / g.

[0157] 4. Comparison 1

[0158] Compared with method 1, the ammonium sulfate fractionation precipitation step (1) was removed to obtain 4.4 g of uricase enzyme powder with a potency of 3210.8 U / g.

[0159] 5. Contrast 2

[0160] Compared with method one, after ammonium sulfate graded precipitation step (1) and before step (2), a cation chromatography step is further added, specifically, EzFsat Diamond SP Mustang cation exchange resin filler is selected and loaded into a chromatography column, and the column is balanced with a balancing solution of 0.02M pH5.5 buffer (NaAc-HAc); the sample containing uricase is loaded at a flow rate of 2mL / min until overloaded, and after loading, it is balanced with a balancing solution (NaAc-HAc) until the conductivity and ultraviolet absorption value are stable; 0.02M pH7.4 PBS containing 0.2M NaCl is used for elution, and the elution peak is collected according to the intensity of the absorption value at 280nm, and then the collected protein is used for dialysis in step (2). The other steps are the same as method one, and finally 2.0g of uricase enzyme powder is obtained, and the titer is 6030.8U / g.

[0161] 6. Contrast 3

[0162] Compared with method 1, step (5) was omitted, and the other steps were the same. The freeze-drying time was 6 hours longer than that of method 1.

[0163] 7. Contrast 4

[0164] The dialysis step in step (2) of method 1 was omitted, and the other steps were the same, and 1.7 g of uricase powder was finally obtained with a potency of 4958.3 U / g.

[0165] 8. Contrast Five

[0166] The dialysis step in step (4) of method 1 was omitted, and the other steps were the same, and 2.7 g of uricase powder was finally obtained with a potency of 4879.4 U / g.

[0167] 9. Compared with method 1, step (3) DEAE cellulose chromatography was omitted, and the other steps were the same. Finally, 3.7 g of uricase enzyme powder was obtained with a titer of 3774.1 U / g.

[0168] 10. Compared with method 1, the PBS buffer in step (3) DEAE cellulose chromatography was replaced with a PBS buffer with a pH of 9.7. The other steps were the same, and finally 2.2 g of uricase powder was obtained with a titer of 3859.5 U / g.

[0169] From the above comparison, it can be seen that the purification method of the present invention can achieve better technical effects, and the potency is greatly improved compared with the comparison method.

Claims

1. A polynucleotide encoding a uricase variant, wherein the polynucleotide is subjected to the following mutations based on the sequence shown in SEQ ID NO.2: positions 541-543 are mutated to TCC and / or positions 544-546 are mutated to AAA. A vector comprising the polynucleotide according to claim 1.

3. A cell comprising the polynucleotide of claim 1 or the vector of claim 2.

4. A method for purifying the uricase variant encoded by the polynucleotide of claim 1, comprising the following steps: (1) Ammonium sulfate graded precipitation Place 100 mL of fermentation liquid containing uricase variants after simple filtration in ice water, slowly add 10.6 g of ammonium sulfate, and stir continuously until the ammonium sulfate is completely dissolved, so that the concentration of ammonium sulfate reaches 20%. Place in ice water bath for 3 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the supernatant; then slowly add 49.7 g of ammonium sulfate to the supernatant, and stir continuously until the ammonium sulfate is completely dissolved, so that the concentration of ammonium sulfate reaches 90%; place in ice water bath for 24 hours, centrifuge at 4°C and 12000 r / min for 15 minutes, and retain the precipitate; then use 20 mL of 1 mM, pH 6.0 PBS to dissolve the precipitate, and store in a refrigerator at 2-8°C; (2) Dialysis The enzyme solution obtained in step (1) was placed in a dialysis bag, which was sealed. The dialysis bag was then placed in a 1 mM, pH 6.0 PBS buffer and dialyzed for 24 h, with the dialysate being changed every 4 h. (3) DEAE cellulose chromatography After the DEAE cellulose chromatography column is equilibrated with 100 mM PBS, pH 7.0, the enzyme solution obtained in step (2) is loaded, and then the column is equilibrated with the PBS, and then eluted with 1 mM PBS buffer containing 0.5 M NaCl and pH 6.0, and the eluted sample is collected; (4) Second dialysis The enzyme solution obtained in step (3) was placed in a dialysis bag, which was sealed. The dialysis bag was then placed in a 1 mM, pH 6.0 PBS buffer and dialyzed for 24 h, with the dialysate being changed every 4 h. (5) Ultrafiltration concentration Performing membrane filtration on the enzyme solution obtained in step (4); (6) Freeze-drying The enzyme solution obtained in step (5) is placed in a -80°C refrigerator and frozen overnight, and then freeze-dried to obtain uricase enzyme powder.

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