CN117230034A

CN117230034A - High-stability mammal urate oxidase mutant

Info

Publication number: CN117230034A
Application number: CN202311336258.4A
Authority: CN
Inventors: 李佳宁; 张淳; 郭勇; 刘璐; 赵志龙
Original assignee: Linyi University
Current assignee: Linyi University
Priority date: 2023-10-16
Filing date: 2023-10-16
Publication date: 2023-12-15

Abstract

According to the evolution trend of the urate oxidase, a mutant library is constructed by analyzing the conserved sequences of various mammal urate oxidases, a high-stability mammal urate oxidase mutant JC is obtained by screening, site-directed mutagenesis is carried out on the mutant JC, and mutation L58V, R291K, A S with higher activity and stability and a combination thereof are further screened. The invention provides a high-stability mammal urate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO:1 or 5. The mutant has a significantly improved stability, e.g., at least 2, 3, 4, 5 or 10-fold improvement in stability, compared to wild-type urate oxidase, wherein the ratio of stability refers to the ratio of retention of specific activity of the enzyme when placed at 25-40 ℃ for over 0.5h, and at the same time has an improved specific activity of the enzyme.

Description

High-stability mammal urate oxidase mutant

Technical Field

The invention relates to the fields of genetic engineering and enzyme engineering, in particular to a high-stability mammal urate oxidase mutant.

Background

In recent years, the incidence rate of hyperuricemia in China has a remarkable rising trend, and the characteristic of younger generation is presented. Statistics show that the total prevalence rate of Chinese hyperuricemia is 13.3 percent, and the prevalence rate is about 1.77 hundred million. Hyperuricemia has risen to the fourth of the common diseases in our country, next to diabetes, hypertension and hyperlipidemia. If not treated in time, gout is gradually evolved, and various complications such as obesity, hypertension, fatty liver, chronic kidney disease, cardiovascular and cerebrovascular diseases and the like are accompanied. Hyperuricemia has become one of the major diseases that pose a serious threat to human health, and the rank is gradually rising. The progression of hyperuricemia and gout is a continuous, chronic pathophysiological process, whose treatment and management requires long-term, and even lifelong, condition monitoring and intervention.

In the process of human evolution, human beings cannot synthesize active uricase due to missense mutation of uricase genes, and uric acid cannot be effectively degraded. In the field of health products, celery seed and sour cherry are considered as effective tools for reducing uric acid levels in vivo. In the uric acid pharmaceutical market, the inactive drug febuxostat tablet is attracting attention. The above method can only reduce deposition of uric acid and cannot have an effect on the tophus that has already formed. Uricase has the ability to catalyze the decomposition of uric acid to 5-hydroxyisouric acid, which is self-hydrolyzed to water-soluble allantoin, which is excreted with urine. Therefore, uricase is widely used in the treatment of hyperuricemia and gout. Uricase drugs have not been marketed in China, and in the International market, U.S. FDA approved Saccharomyces cerevisiae recombinant Aspergillus flavus-derived uricase (Rasburicase) in 2002. Because of the large difference between the polypeptide and the humanized uricase, the homology is lower than 40%, and the polypeptide is easy to induce immune response in vivo, and can only be used for short-term treatment of acute hyperuricemia. Polyethylene glycol (PEG) modified escherichia coli recombinant swine-derived uricase (Pegloticase) approved by the FDA in 2010, although it is a PEG modified long-acting protein drug from mammals, has the unique efficacy of rapidly ablating tophus, and is considered as a potential breakthrough drug for treating refractory gout (sold in 2019 for 3.4 billion dollars). However, the drug has the defects of high immunogenicity, reduced effective rate after long-term injection and the like.

Primates with high homology to human uricase, while capable of producing uricase, have extremely limited activity and lack research value. The homology of other mammal-derived uricase and human-derived uricase exceeds 90%, and the method becomes an important research direction for developing long-acting uricase medicines. However, mammalian uricase activity is generally low and exhibits poor stability in complex physiological environments in humans. Such low activity and instability leads to increased doses and times of drug injections, and excessive injections may lead to immune responses in the patient. Therefore, it is important to construct uricase of mammalian origin with high stability, which can reduce the injection times and dosage of the medicine, so that the uricase can maintain the activity state in human body for a long time and continuously decompose uric acid. In view of the above, the development of highly active and highly stable mammalian uricase is one of the key strategies for developing highly safe, low-immunogenicity, long-acting uricase drugs.

Patent (WO 2019/010369) discloses recombinant mutant candida utilis (candida utilis) urate oxidase with improved pancreatin stability and/or activity; patent (WO 2021/068925) discloses an improved Micrococcus (Arthrobacter globiformis) urate oxidase with improved enzymatic activity and thermostability over the original structure. The urate oxidase proteins of the above patents are all of microbial origin, non-mammalian origin, and may present a risk of too high immunogenicity similar to Aspergillus flavus-derived urate oxidase (Rasburicase) when applied to humans. The patent (WO 2011/050599) discloses humanized recombinant urate oxidase and mutants thereof, the patent (CN 104630168A) discloses a chimeric urate oxidase structure of a human pig, and the humanized degree is improved mainly through chimeric, so that high-activity and high-stability urate oxidase screening is not involved. The methods disclosed in the prior art are not directed to further enhancing enzyme activity and stability based on mammalian urate oxidase structure.

Disclosure of Invention

In order to solve the technical problems, the invention provides a high-stability mammal urate oxidase (also simply called uricase) mutant, wherein the stability of the mutant is obviously improved compared with that of wild type urate oxidase (for example, the stability is improved by 2 times compared with that of wild type canine urate oxidase).

The invention provides a high-stability mammal urate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO:1 or with SEQ ID NO:1 comprising the mutation L58V, R291K, A S and combinations thereof.

In some embodiments, the mutant may also comprise other suitable mutations, provided that the mutation does not substantially reduce stability and enzyme specific activity. In some embodiments, the mutant hybridizes to a polypeptide as set forth in SEQ ID NO:3, wherein the stability is increased by at least 2, 3, 4, 5 or 10 times as compared with the wild-type canine urate oxidase, and the ratio of stability is the ratio of the retention rate of specific activity of the enzyme which is placed at 25-40 ℃ for more than 0.5h, for example, 0.5h, 1h, 2h or 3h at 37 ℃.

In some embodiments, the amino acid sequence of the mutant is as set forth in SEQ ID NO:1 or 5.

The invention also provides a nucleic acid molecule comprising the coding sequence of a high stability mammalian urate oxidase mutant according to any of the embodiments of the invention; preferably, the nucleic acid molecule is DNA or RNA.

The invention also provides a gene expression frame, which comprises a promoter and a coding sequence operably connected behind the promoter, wherein the coding sequence is the coding sequence of the high-stability mammal urate oxidase mutant according to any scheme of the invention.

In some embodiments, the promoter is selected from the group consisting of a lactose promoter system, a tryptophan promoter system, a beta lactamase promoter system, or a promoter system derived from phage lambda or T7.

In some embodiments, the nucleotide sequence of the coding sequence is set forth in SEQ ID NO:2 or 4.

The invention also provides an expression vector comprising the gene expression cassette according to any one of the schemes of the invention.

The present invention provides a host cell comprising a nucleic acid molecule, gene expression cassette or expression vector according to any one of the embodiments of the present invention.

In some embodiments, the host cell is selected from a mammalian cell, an insect, a yeast, or a bacterial cell; preferably an enterobacter cell or a yeast cell.

The invention also provides a method for producing a high stability mammal urate oxidase mutant according to any one of the embodiments of the invention, comprising: expressing the mutant in the host cell, and separating and purifying.

The invention also provides application of the high-stability mammal urate oxidase mutant in preparing urate oxidase medicaments.

Compared with the prior art, the invention has the beneficial effects that at least:

(1) According to the evolution trend of the urate oxidase, the invention constructs a mammal urate oxidase mutant library by analyzing the conserved sequences of various mammal urate oxidases, and screens to obtain the high-stability mammal urate oxidase mutant JC.

(2) The mutant JC is subjected to site-directed mutagenesis, the mutant L58V, R291K, A S with higher activity and stability and the combination thereof are further screened, and compared with the wild type canine urate oxidase with the highest activity, the mutant JC has at least 2 times of stability and higher enzyme specific activity.

Drawings

FIG. 1 is an alignment of 22 mammalian-derived uricase sequences.

FIG. 2 is an identification of purity of a mammalian-derived uricase mutant protein. A: SDS-PAGE identification. M: a high molecular weight pre-dye protein marker;1: JC;2: JC (JC) _{L58V-R291K-A301S} . And B, HPLC identification.

FIG. 3 is a comparison of the enzymatic activity of highly stable mammalian urate oxidase mutants compared to wild-type canine urate oxidase.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. Examples of which are illustrated in the accompanying drawings. It should be understood that the specific examples described in the following embodiments of the present invention are intended to be illustrative of the specific embodiments of the present invention and are not to be construed as limiting the invention.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass the range or value as being close to the range.

The invention provides a high-stability mammal urate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1.

Through systematic studies, the inventor discovers that as evolution progresses, mammal urate oxidase of various species has a trend of lower and lower enzyme activity. By comparing with multiple sequences of mammal urate oxidase with high homology of human urate oxidase, amino acid sequences with high conservation or commonality are selected, missense mutation accumulated in the evolution process of single species mammal urate oxidase can be eliminated, and mammal urate oxidase mutant protein with higher activity can be obtained.

Based on the protein sequences of mammal urate oxidase of dogs, pigs, cattle, rabbits, rats, night monkeys and the like, the wild mammal urate oxidase has been proved to have the activity of degrading uric acid by the inventor, and the total length of the urate oxidase protein sequence is 300-304 amino acids; the sequence Identity (Identity) between the wild-type mammal urate oxidase and the deduced human urate oxidase should be not less than 85.0% by software analysis such as Blast. Based on 22 or more amino acid sequences of mammal urate oxidase, the highly conserved region is reserved through multi-species sequence Alignment (Alignment), and 1 or more alternative amino acid compositions are designed in a non-conserved variable region through consensus analysis.

More specifically, when the amino acid sequence difference of the variable region among multiple species is large, firstly 1-2 amino acids with the highest conservation or commonality are selected, for example, at 52 th site, canine, porcine, rabbit, night monkey, artificial serine, bovine asparagine, rat arginine, serine commonality is obviously higher than other two amino acids, and serine with the highest commonality is selected at 52 th site; if arginine is taken as canine, bovine and porcine sources at 291, and the shareability of arginine and lysine is not obviously different among rabbits, rats, night monkeys and artificial lysine, arginine and lysine are respectively selected at 291, and two alternative mutant sequences are constructed. By analogy, 1 or more amino acids with highest commonality are selected at amino acid positions with sequence differences, and a mammalian urate oxidase mutant library is constructed therefrom. Obtaining JC protein amino acid sequence with highest conservation or commonality of the mammal urate oxidase of multiple species, namely the amino acid sequence shown as SEQ ID NO:1, and a highly stable mammalian urate oxidase mutant as shown in the formula 1.

In order to further increase the activity and thermostability, the invention also provides SEQ ID NO:1, and constructing different mammal urate oxidase mutant DNA sequences by means of site-directed mutagenesis. The site-directed mutagenesis method can be achieved by various techniques known to those skilled in the art, such as DNA recombination, PCR, etc., including but not limited to the two-round staggered-extension PCR method and the site-directed mutagenesis method described in quick change of Strantagene.

Further, in the sequence set forth in SEQ ID NO:1, and the mutation L58V, R291K, A S and the combination thereof can improve the specific activity or stability of the enzyme or not obviously reduce the stability on the basis of improving the specific activity of the enzyme. The present invention thus also provides highly stable mammalian urate oxidase mutants which are identical to the amino acid sequence of SEQ ID NO:1 comprising the mutation L58V, R291K, A S and combinations thereof. L58V refers to SEQ ID NO: leucine at position 58 of 1 to valine; R291K is SEQ ID NO:1 to lysine; a301S refers to SEQ ID NO:1 to serine.

In some embodiments, the mutant may also comprise other suitable mutations, provided that the mutation does not substantially reduce stability and enzyme specific activity. In some embodiments, the mutant hybridizes to a polypeptide as set forth in SEQ ID NO:3, wherein the stability ratio is at least 2, 3, 4, 5 or 10 times higher than that of wild-type canine urate oxidase, and the ratio of stability ratio is that the retention rate of specific activity of enzyme is higher than 0.5h when the wild-type canine urate oxidase is placed at 25-40 ℃, for example, the wild-type canine urate oxidase is placed at 37 ℃ for 0.5h, 1h, 2h or 3h. As shown in example 6, the stability was improved by about 2.91 times when left at 37℃for 1 hour, the wcu enzyme activity retention was 31.75%, and the JC enzyme activity retention was 92.40%.

In some embodiments, the amino acid sequence of the mutant is as set forth in SEQ ID NO:1 or 5, wherein the sequence of the mutant is shown as SEQ ID NO:1 or both the mutations L58V, R291K and a301S.

According to the highly stable mammalian urate oxidase mutant, the amino acid sequence may synthesize a corresponding DNA sequence or mRNA sequence for use in producing the urate oxidase mutant. The invention also provides a nucleic acid molecule comprising a sequence encoding a high stability mammalian urate oxidase mutant according to any of the aspects of the invention; preferably, the nucleic acid molecule is DNA or RNA. When the nucleic acid molecule is DNA, synthesizing the urate oxidase mutant through translation expression of the DNA, and using the urate oxidase mutant for protein production; the RNA may be mRNA for transient expression in a cell.

The invention also provides a gene expression cassette, which comprises a promoter and a coding sequence operably linked to the promoter, wherein the coding sequence codes for a high-stability mammal urate oxidase mutant sequence according to any scheme of the invention.

By the correspondence of amino acids to nucleotides, and codon optimization, one skilled in the art can obtain different nucleotide sequences. In some embodiments, the nucleotide sequence of the coding sequence is set forth in SEQ ID NO:2 or 4.

The invention also provides an expression vector comprising the gene expression cassette according to any one of the schemes of the invention. The expression vector is preferably a plasmid vector, which can be expressed in mammalian cells, insects, yeasts, bacteria or other cells by cell transduction.

Thus, the present invention provides a host cell comprising a nucleic acid molecule, gene expression cassette or expression vector according to any one of the embodiments of the present invention.

The mammalian urate oxidase mutant protein can be isolated from the inside or outside of host cell (such as culture medium) and purified to obtain high purity homogeneous protein. The method for separating and purifying the protein is not limited to any particular method, such as column chromatography, filtration, ultrafiltration, salting-out, isoelectric precipitation, dialysis, etc. For chromatography, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel exclusion chromatography, reverse phase chromatography, etc., may be applied. These chromatography may be performed using liquid chromatography such as a rapid protein liquid chromatography system. The protein purity and concentration are detected by a general protein detection method, such as HPLC method, SDS-polyacrylamide electrophoresis method, isoelectric point electrophoresis method, BCA method, lowry method, kjeldahl nitrogen method, etc. The specific activity of urate oxidase is detected by uric acid substrate degradation method, for example, based on the characteristic absorption peak of substrate uric acid at 293nm, the uric acid consumption rate is detected by ultraviolet spectrophotometry or high performance liquid chromatography, and the urate oxidase activity per unit volume is calculated. The specific activity of the urate oxidase protein is calculated by the monomer volume urate oxidase activity and the unit volume urate oxidase protein concentration. The thermal stability of the urate oxidase is detected by a thermal stability detection method at 25-37 ℃. More preferably, the thermal stability of the urate oxidase is detected by a method such as 37 ℃ thermal stability detection.

The invention also provides application of the high-stability mammal urate oxidase mutant in preparing urate oxidase medicaments. The urate oxidase medicament can be used for treating hyperuricemia and gout.

Example 1 multiple species mammalian urate oxidase bioinformatics analysis and initial mutant design

Based on the translation of the sequence of the pseudogene of human uricase (GenBank: AB 074326.2) into an amino acid sequence, nonsense mutations (nonenseuntion) at positions 33 and 187 were replaced with arginine and arginine, respectively, of the night monkey uricase. And (3) performing Blast analysis on the protein sequence to obtain the amino acid sequence of the mammal uricase with the Identity of not less than 85%. The amino acid sequence of the mammal uricase is subjected to multi-sequence Alignment analysis (shown in figure 1) by using Clustalomega and other software, and JC protein amino acid sequences with the highest conservation or commonality of the mammal uricase of multiple species are obtained as follows:

MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLASRL(SEQ ID NO：1)。

according to the amino acid sequence, DNA design is carried out according to the preferred codons of escherichia coli and saccharomycetes, and corresponding companies are entrusted to carry out total gene synthesis, wherein the DNA sequence corresponds to:

CATATGGCCCATTATCATAATGATTATAAAAAAAATGATGAAGTTGAATTTGTTCGTACCGGTTATGGTAAAGATATGGTTAAAGTTCTGCATATTCAGCGTGATGGTAAATATCATTCTATTAAAGAAGTTGCCACCTCTGTTCAGCTGACCCTGTCTTCTAAAAAAGATTATCTGCATGGTGATAATTCTGATATTATTCCAACCGATACCATTAAAAATACCGTTCATGTTCTGGCCAAATTTAAAGGTATTAAATCTATTGAAACCTTTGCCATGAATATTTGTGAACATTTTCTGTCTTCTTTTAATCATGTTATTCGTGCCCAGGTTTATGTTGAAGAAGTTCCATGGAAACGTTTTGAAAAAAATGGTGTTAAACATGTTCATGCCTTTATTCATACCCCAACCGGTACCCATTTTTGTGAAGTTGAACAGCTGCGTTCTGGTCCACCAGTTATTCATTCTGGTATTAAAGATCTGAAAGTTCTGAAAACCACCCAGTCTGGTTTTGAAGGTTTTATTAAAGATCAGTTTACCACCCTGCCAGAAGTTAAAGATCGTTGTTTTGCCACCCAGGTTTATTGTAAATGGCGTTATCATCAGGGTCGTGATGTTGATTTTGAAGCCACCTGGGATACCGTTCGTGATATTGTTCTGGAAAAATTTGCCGGTCCTTATGATAAAGGTGAATATTCTCCATCTGTTCAGAAAACCCTGTATGATATTCAGGTTCTGTCTCTGTCTCGTGTTCCAGAAATTGAAGATATGGAAATTTCTCTGCCAAATATTCATTATTTTAATATTGATATGTCTAAAATGGGTCTGATTAATAAAGAAGAAGTTCTGCTGCCACTGGATAATCCTTATGGTCGTATTACCGGTACCGTTAAACGTAAACTGGCCTCTCGTCTGTGATAAGGATCC(SEQ ID NO：2)。

the recombinant plasmid obtained by total gene synthesis is amplified, then cut by NdeI and BamHI double enzyme cutting method, and then the needed target fragment is recovered. The plasmid pET-3C (Invitrogen), which was also digested with NdeI and BamHI, was ligated to the cut target fragment by T4DNA ligase. The ligated mixture was then transformed into E.coli clone host strain TOP10 according to the standard procedure described in Current Protocols in Molecular Biology.

Transformation reactions were performed on LB plates containing ampicillin, and after overnight incubation, transformed monoclonal colonies were selected. These colonies were screened by means of digestion and PCR validation to prepare strains containing recombinant plasmid pET-3C-JC. Through DNA sequencing test, the JC sequence in the positive recombinant plasmid is ensured to be completely consistent with the theoretical sequence.

EXAMPLE 2 recombinant expression of uric acid oxidase in multiple species of mammals

The correctly sequenced JC recombinant plasmid was transformed for expression in E.coli host bacteria. JC proteins were expressed using these strains of E.coli BL21 (DE 3), BL21Star (DE 3) or BL21Star (DE 3) plysS. These strains are one of many suitable strains for expression of chimeric proteins, available commercially from Novagen, invitrogen and Stratagen, respectively. The transformed cells were cultured on LB plates containing ampicillin, and correct colonies were identified by their growth ability.

Coli containing the JC recombinant plasmid was cultured overnight in 50ug/ml ampicillin in liquid LB medium. Subsequently, the overnight cultures were inoculated into large cultures at a ratio of 1:100. When the optical density of the cells reaches a specific value (typically 600 nm), IPTG is added to a final concentration of 0.5mM to induce expression of the protein of interest. The cells were further cultured for 3 hours, collected by centrifugation, the cell pellet was washed with 50mM Tris buffer, and stored at-20 ℃. Detection of protein expression by SDS-PAGE ensures successful expression of recombinant proteins.

EXAMPLE 3 Multispecies mammalian urate oxidase mutant library design

A series of mutation sites are designed according to the degree of commonality by taking JC protein DNA sequence as an original template, for example: L58V (mutation is expressed by triplet: letter-number-letter, wherein the number indicates the position of the mutated amino acid, the letter before the number corresponds to the amino acid designed for mutation, the letter after the number indicates the amino acid used for replacing the amino acid before the number, the numbers are ordered according to amino acid 304 of human uricase), Q109HL146M, S148N, T35213A, S246T, V250L, A S.

Specifically, based on JC protein, mutant protein with at least one point mutation is prepared by using a staggered extension PCR method to construct a mutant library. Then, recombinant expression, preparation and enzyme specific activity detection were performed according to the methods in examples 3, 4, and 5. Through this process, useful mutations that have an increased specific activity of the enzyme without detrimental effects are selected, from which the most stable mammalian uricase mutants are further selected. In this process, the inventors have found through extensive experiments that the enzyme specific activity and stability of wild-type canine uricase protein are relatively high (named wCU, SEQ ID NO: 3), so that uricase mutants in this study were compared with recombinant wild-type canine uricase protein.

The amino acid sequence of wild-type canine uricase is as follows:

MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSV

QLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSF

NHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIH

SGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVD

FEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEIS

LPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTAKRKLASKL(SEQ ID NO：3)。

the DNA containing the target mutation was prepared by mutation using the staggered-extension PCR method. The preparation of L58V, R291K-A301S and L58V-R291K-A301S (SEQ ID NO: 5) is described below as examples:

primer numbering	DNA sequence
		Primer 1 (SEQ ID NO: 6)	5'CACGACATATGGCCCATTATCATA3'
Primer 2 (SEQ ID NO: 7)	5'GGATCCTTATCACAGACGAGAGGC3'
		Primer 3 (SEQ ID NO: 8)	5'GATTATGTTCATGGTGATAAT3'
Primer 4 (SEQ ID NO: 9)	5'ACCATGAACATAATCTTTTTT3'
		Primer 5 (SEQ ID NO: 10)	5'GGATCCTTATCACAGACGAGAGCTCAGTTTACG3'

Preparation of JC _L58V : the first stage PCR is that the template sequence is the total gene synthesis sequence (SEQ ID NO: 2) in example 1, the primers are primer 1 and primer 3 in the table, and the PCR reaction system and method are all set by adopting a PCR reaction kit according to the instruction of a merchant. The PCR reaction conditions were: 94℃1min,56℃1min,72℃1min, 30 cycles, the first cycle denaturation at 94℃for 10min, the last cycle extension at 72℃for 10min. Amplified by the PCR conditions to obtain the product JC _L58V -a fragment; second stage PCR: the template sequence is the same with the first stage PCR, the primer is primer 2 and primer 4, and JC is obtained by amplifying according to the PCR conditions _L58V -fragment b; third stage PCR: JC template _L58V Fragment-a and JC _L58V -1 of fragment b: 1, the primer is primer 1 and primer 2, and the product JC is obtained by amplifying the mixture solution according to the PCR conditions _L58V . The DNA sequence containing the mutated sequence was digested simultaneously with NdeI and BamHI, ligated, transformed, screened and expressed as in example 2.

Preparation of JC _R291K-A301S : because the mutation position is at the C end of uricase, the primer 1 and the primer 5 are directly utilized, the complete gene synthesis sequence (SEQ ID NO: 2) in the example 1 is taken as a template, and the PCR condition is used for amplification to obtain a product JC _R291K-A301S 。

Preparation of JC _{L58V-R291K-A301S} : according to the site-directed mutagenesis method, after the 58 th mutation is finished, the 301 st amino acid mutation is carried out by taking the 58 th mutation as a template, and the PCR conditions and the PCR methods are the same.

EXAMPLE 4 purification of mammalian uricase mutant expression

Adding 50g of thallus sediment into 500ml of bacteria breaking liquid with pH of 8.3 and 25mM Tris-HCl,5mMEDTA,0.1mg/ml lysozyme, stirring at 37 ℃ for 60-80 minutes,then 1mM MgCl was added ₂ ·6H ₂ O、1μg·mL ^-1 Stirring the nuclease overnight; 8500 r.min at 4 ℃ ^-1 Centrifuging for 20min, and collecting bacterial precipitate. Taking a bacterial breaking precipitate, and mixing the bacterial breaking precipitate with 100g of wet bacteria: 1L of a washing solution (25 mM Tris-HCl,5mM EDTA, 1.5% Triton X-100, pH 8.3) was suspended and precipitated, and after homogenization, stirred at 30℃for 2 hours; 8500 r.min ^-1 Centrifuging at 4deg.C for 15min, collecting precipitate, washing with the same washing solution, and centrifuging to collect precipitate. Taking TritonX-100, washing the precipitate, and preparing 100g of wet bacteria: 1L of the washing solution (PBS, 0.34. Mu.g.multidot.mL) ^-1 Nuclease) suspended sediment, stirring for 2 hours at 30 ℃, centrifuging for 15 minutes at 8500 r.min < -1 >, and centrifuging for collecting sediment. The pellet was washed with PBS and nuclease, still at 100 grams wet: 1 liter of washing solution (PBS) is suspended and precipitated, homogenized by a homogenizer, stirred at 30 ℃ for 2 hours, 8500 r.min ^-1 Centrifuging at 4deg.C for 15min, collecting precipitate, washing with the same washing solution, and centrifuging to collect precipitate. Taking a precipitate after bacterial breaking washing, and precipitating according to 10 g: 3L lysis buffer (0.1 MNA ₂ CO ₃ -NaHCO ₃ pH10.3), and after homogenization with a homogenizer, stirring overnight at room temperature. 8500 r.min ^-1 Centrifuging at 4 ℃ for 15min, and collecting supernatant. Adding 10% saturated (NH 4) to the supernatant ₂ SO ₄ Standing overnight at 4 ℃ for 8500 r.min ^-1 Centrifuging at 4deg.C for 15min, and collecting the centrifugal precipitate; dissolving buffer solution, stirring overnight at room temperature, 8500 r.min ^-1 Centrifuging at 4 ℃ for 15min, and collecting supernatant. Purifying the supernatant by DEAE agarose anion exchange chromatography (GE), and subjecting the whole target protein to column chromatography with 0-0.2M NaCl (pH 10.30.1 MNA) ₂ CO ₃ -NaHCO ₃ ) Eluting, wherein the target protein is eluted at the time of 0.1 MNaCl; concentrating the eluate by DEAE Sepharose anion exchange chromatography column (GE), and purifying with chromatography column (pH 10.30.1 Mna) containing 0.2MNaCl ₂ CO ₃ -NaHCO ₃ ) The eluent elutes the target protein. At this time, purity was detected by SDS-PAGE and HPLC and was found to be 95% or more (as shown in FIG. 2).

EXAMPLE 5 detection of mammalian uricase mutant protein Activity

At 37℃and pH8.6, 1. Mu. Mol of uric acid was converted per minuteThe amount of enzyme that is allantoin is defined as one International Unit (IU). Uric acid has characteristic absorption peak at 293nm, when it is degraded by uricase, the product has no absorption peak in this wavelength range, the change of absorbance at 293nm is detected at regular time to determine the decrease of uric acid, and then the molar extinction coefficient (1.23×104M is used ^-1 ·CM ^-1 ) The uric acid concentration is calculated, and the uricase activity can be calculated from the change in uric acid concentration. After the ultraviolet spectrophotometer is regulated to 293nm and the stand-by device is stabilized, the blank is zeroed by using 0.1M sodium tetraborate solution, 3ml of 0.1mM uric acid solution is taken for reaction and dissolution, and then the solution is added into a quartz cuvette, 10ul uricase mutant protein is supplemented, reading is carried out every 30 seconds, and the OD293 change value within 2 minutes is measured. According to the formula C=A/Kb (C is the uric acid concentration of the solution, A is the light absorption value of 293nm, K is the molar extinction coefficient-1.23×104M) ^-1 ·CM ^-1 B is the inner diameter of the cuvette), calculate the OD at different time points ₂₉₃ Corresponding uric acid concentrations; calculating the reduced uric acid substance amount from Δm= Δcv (Δm is the number of moles of uric acid reduction, Δc is the change in uric acid concentration, C is the reaction liquid volume); uricase activity was calculated from U= [ delta ] M/TV1 (U is uricase activity unit contained in per milliliter of plasma, T is reaction minutes, V1 is uricase mutant protein volume added to the reaction system) (as shown in FIG. 3), and the results showed JC and JC _{L58V-R291K-A301S} The specific activity of (2) is 7.13% and 14.11% higher than that of wild-type canine uricase, respectively.

EXAMPLE 6 mammalian uricase mutant thermal stability assay

Uricase mutant proteins were buffered with buffer (0.1 MNA ₂ CO ₃ -NaHCO ₃ pH10.3), diluted to 4mg/ml, placed in a 37 ℃ incubator, after 0h, 0.5h, 1h, 2h, 3h, respectively, and taken out of the sample, according to example 5 method to determine uricase activity, comparison of enzyme activity retention.

The results show JC and JC _{L58V-R291K-A301S} The thermal stability of uricase protein is obviously better than that of wild canine uric acidEnzymes which retain 79.72% and 35.50% activity after 3h at 37℃are approximately 19.35 and 8.50 times the wild-type activity, respectively.

Other sequences to which the examples relate

JC _{L58V-R291K-A301S} Is a DNA sequence of (2):

CATATGGCCCATTATCATAATGATTATAAAAAAAATGATGAAGTTGAATTTGTTCGTACCGGTTATGGTAAAGATATGGTTAAAGTTCTGCATATTCAGCGTGATGGTAAATATCATTCTATTAAAGAAGTTGCCACCTCTGTTCAGCTGACCCTGTCTTCTAAAAAAGATTATGTTCATGGTGATAATTCTGATATTATTCCAACCGATACCATTAAAAATACCGTTCATGTTCTGGCCAAATTTAAAGGTATTAAATCTATTGAAACCTTTGCCATGAATATTTGTGAACATTTTCTGTCTTCTTTTAATCATGTTATTCGTGCCCAGGTTTATGTTGAAGAAGTTCCATGGAAACGTTTTGAAAAAAATGGTGTTAAACATGTTCATGCCTTTATTCATACCCCAACCGGTACCCATTTTTGTGAAGTTGAACAGCTGCGTTCTGGTCCACCAGTTATTCATTCTGGTATTAAAGATCTGAAAGTTCTGAAAACCACCCAGTCTGGTTTTGAAGGTTTTATTAAAGATCAGTTTACCACCCTGCCAGAAGTTAAAGATCGTTGTTTTGCCACCCAGGTTTATTGTAAATGGCGTTATCATCAGGGTCGTGATGTTGATTTTGAAGCCACCTGGGATACCGTTCGTGATATTGTTCTGGAAAAATTTGCCGGTCCTTATGATAAAGGTGAATATTCTCCATCTGTTCAGAAAACCCTGTATGATATTCAGGTTCTGTCTCTGTCTCGTGTTCCAGAAATTGAAGATATGGAAATTTCTCTGCCAAATATTCATTATTTTAATATTGATATGTCTAAAATGGGTCTGATTAATAAAGAAGAAGTTCTGCTGCCACTGGATAATCCTTATGGTAAAATTACCGGTACCGTTAAACGTAAACTGAGCTCTCGTCTGTGATAAGGATCC(SEQ ID NO：4)。

the corresponding amino acid sequence:

MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVHGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO：5)。

finally, the above embodiments are only for illustrating the technical solution of the present invention, and do not limit the present invention. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims

1. A high-stability mammal urate oxidase mutant, which is characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO:1 or with SEQ ID NO:1 comprising the mutation L58V, R291K, A S and combinations thereof.

2. The mutant of claim 1, wherein the mutant hybridizes to a sequence set forth in SEQ ID NO:3, wherein the stability is improved by at least 2, 3, 4, 5 or 10 times compared with the wild-type canine urate oxidase, and the ratio of the stability is the ratio of the retention rate of the specific activity of the enzyme which is placed at 25-40 ℃ for more than 0.5 h.

3. The mutant of claim 1, wherein the amino acid sequence of the mutant is as set forth in SEQ ID NO:1 or 5.

4. A nucleic acid molecule comprising the coding sequence of the high stability mammalian urate oxidase mutant according to any one of claims 1 to 3; preferably, the nucleic acid molecule is DNA or RNA; more preferably, the nucleotide sequence of the coding sequence is as set forth in SEQ ID NO:2 or 4.

5. A gene expression cassette comprising a promoter and a coding sequence operably linked to the promoter, wherein the coding sequence is that of a high stability mammalian urate oxidase mutant according to any one of claims 1 to 3.

6. The gene expression cassette of claim 5, wherein the promoter is selected from the group consisting of lactose promoter system, tryptophan promoter system, beta lactamase promoter system, and phage lambda or T7 derived promoter system; the nucleotide sequence of the coding sequence is shown as SEQ ID NO: 2.4 or 6.

7. An expression vector comprising the gene expression cassette of claim 5 or 6.

8. A host cell comprising the nucleic acid molecule of claim 4, the gene expression cassette of claim 5 or 6, or the expression vector of claim 7; preferably, the host cell is selected from mammalian cells, insect, yeast or bacterial cells; more preferably an enterobacter cell or a yeast cell.

9. A method of producing a high stability mammalian urate oxidase mutant according to any one of claims 1 to 3 comprising: expressing the mutant in the host cell, and separating and purifying.

10. Use of a highly stable mammalian urate oxidase mutant according to any one of claims 1 to 3 for the preparation of a urate oxidase drug.