CN117327673A - A highly active mammalian urate oxidase mutant - Google Patents

Abstract

Based on the evolutionary trend of urate oxidase, the present invention constructs a mutant library by analyzing the conserved sequences of multiple mammalian urate oxidases, screens to obtain a highly active mammalian urate oxidase mutant MU, and then performs site-directed mutation on the mutant MU. The mutations E208D and R249Q with higher activity were further screened. The present invention provides a highly active mammalian urate oxidase mutant, the amino acid sequence of which is shown in any one of SEQ ID NO: 1, 5 or 7. The enzyme specific activity of the mutant is significantly improved compared to wild-type urate oxidase. For example, the enzyme specific activity is at least 25% higher than that of wild-type canine urate oxidase, and has higher thermal stability.

Description

A highly active mammalian urate oxidase mutant

Technical field

The invention relates to the fields of genetic engineering and enzyme engineering, and specifically relates to a highly active mammalian urate oxidase mutant.

Background technique

Hyperuricemia and gout are major diseases that seriously endanger human health. Existing therapeutic drugs can only control the onset and continued development of this type of disease from the aspects of anti-inflammatory, analgesic and inhibiting the production of uric acid, but they cannot achieve the purpose of radical cure. Uric acid oxidase drugs can hydrolyze slightly soluble uric acid into easily soluble allantoin, and have been used in the treatment of hyperuricemia and gout caused by tumor radiotherapy and chemotherapy and metabolic disorders. Currently, there are no urate oxidase drugs on the market in China. The internationally marketed urate oxidase drugs include brewer's yeast recombinant aflatoxin-derived urate oxidase (Rasburicase) approved by the US FDA in 2002 and polyethylene glycol approved by the FDA in 2010. (PEG) modified Escherichia coli recombinant porcine urate oxidase (Pegloticase). The former has low homology (<40%) with the presumed human urate oxidase, and antibodies are easily produced in subjects after multiple administrations, so it can only be used for the short-term treatment of acute hyperuricemia; the latter It is a PEG-modified long-acting biological protein drug that can be injected once every two weeks. It has the unique effect of rapidly ablating tophi. It is hailed as a potential breakthrough treatment drug for refractory gout (Pelgoticase sales in 2019 were US$340 million), but there are It has shortcomings such as high immunogenicity and low long-term injection effectiveness.

Mammalian urate oxidase is an important source for the development of long-acting urate oxidase drugs due to its high homology (>90% homology with human urate oxidase). However, the activity of mammalian urate oxidase is low, and low activity means high dosage. The injection dosage is too high (8.0 mg/dose), which is an important factor leading to the excessive immunogenicity of Pelgoticase (PEG-modified porcine recombinant urate oxidase). one. Constructing high-specific activity mammalian-derived urate oxidase and reducing the injection dose are important means to develop low-immunogenic urate oxidase. At the same time, the injection period of PEG-modified urate oxidase drugs is about 2 weeks, which improves the stability of urate oxidase protein, prolongs the metabolism rate in the body, and can also reduce the initial injection dose, further reducing the potential immunogenicity of mammalian urate oxidase drugs. . Developing mammalian-derived urate oxidase with high activity and stability is an important means to develop long-acting urate oxidase drugs with high safety and low immunogenicity.

The patent (WO2019/010369) discloses a recombinant mutant Candida utilis urate oxidase with improved trypsin stability and/or activity; the patent (WO2021/068925) discloses an improved Micrococcus genus (Arthrobacter globiformis) urate oxidase, the enzyme activity and thermal stability are improved compared with the original structure. The above-mentioned patented urate oxidase proteins are all of microbial origin, not mammalian origin. When used in humans, there may be a risk of excessive immunogenicity similar to Aspergillus aflatoxin-derived urate oxidase (Rasburicase). The patent (WO 2011/050599) publishes humanized recombinant urate oxidase and its mutants, and the patent (CN104630168A) publishes the structure of human-pig chimeric urate oxidase. Both of the above two patents mainly improve the degree of humanization through chimerism. , did not involve the screening of high activity and high stability urate oxidase. The methods disclosed in the prior art do not involve further improving enzyme activity and stability based on the structure of mammalian urate oxidase.

Contents of the invention

In order to solve the above technical problems, the present invention provides a highly active mammalian urate oxidase (also referred to as uricase) mutant, the enzyme specific activity of the mutant is significantly improved compared to wild-type urate oxidase ( For example, the enzyme specific activity is approximately 28.9% higher than that of wild-type canine urate oxidase).

The present invention provides a highly active mammalian urate oxidase mutant, the amino acid sequence of the mutant is as shown in SEQ ID NO: 1, or includes mutations E208D and/or R249Q compared with SEQ ID NO: 1.

In some embodiments, the mutant may also include other suitable mutations, as long as the mutations do not significantly reduce the enzyme specific activity. In some embodiments, the mutant has an enzyme specific activity increased by at least 25% compared to wild-type canine urate oxidase as shown in SEQ ID NO: 3.

In some embodiments, the amino acid sequence of the mutant is as shown in any one of SEQ ID NO: 1, 5 or 7.

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

The present invention also provides a gene expression cassette, which contains a promoter and a coding sequence operably connected behind the promoter, and the coding sequence is a high-density protein according to any aspect of the present invention. Coding sequences of active mammalian urate oxidase mutants.

In some embodiments, the promoter is selected from 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 as set forth in any of SEQ ID NO: 2, 4, or 6.

The present invention also provides an expression vector, which includes the gene expression cassette described in any aspect of the present invention.

The present invention provides a host cell, which contains the nucleic acid molecule, gene expression cassette or expression vector described in any aspect of the present invention.

In some embodiments, the host cells are selected from mammalian cells, insect, yeast or bacterial cells; preferably Enterobacteriaceae cells or yeast cells.

The present invention also provides a method for producing a highly active mammalian urate oxidase mutant according to any aspect of the present invention, including: expressing the mutant in the host cell, and isolating and purifying the mutant.

The invention also provides the use of the highly active mammalian urate oxidase mutant in the preparation of urate oxidase drugs.

Compared with the prior art, the beneficial effects of the present invention at least include:

(1) Based on the evolutionary trend of urate oxidase, the present invention constructs a mammalian urate oxidase mutant library by analyzing the conserved sequences of various mammalian urate oxidases, and screens to obtain the highly active mammalian urate oxidase mutant MU.

(2) The present invention then conducts site-directed mutagenesis on the mutant MU, and further screens out the mutations E208D and R249Q with higher activity. Compared with the highest active wild-type canine-derived urate oxidase, the enzyme specific activity is increased by at least 25%, and has Higher thermal stability.

Description of drawings

Figure 1 shows the sequence alignment of different mammalian urate oxidases. A0A5E4C3I6: Woodchuck; P11645: Rabbit; A0A2Y9P065: Beluga whale; P16164: Pig; Q3MHG7: Bovine; A0A6J0XAT6: Odocoileusvirginianus; A0A452G3H7: Goat; W5PWL1: Sheep; P25688: Mouse; P09118: Rat; L9KW26: Tupaia; A0A1S3WDJ8: Erinaceus; P25689: Hamadryas baboon; Q8MKJ2: Aotus; A0A7J7ZXM4: Myotis; G1M4M8: Ailuropoda; :Mustelafuro;A0A2Y9IZI3:Enhydra lutris;A0A673TVJ5:Suricata; A0A485N2I1:Lynx; Q5FZI9:Canine.

Figure 2 shows the recombinant expression identification of mammalian urate oxidase. M: high molecular weight pre-stained protein marker; 1: before induction; 2: after induction.

Figure 3 shows the purity identification of mammalian urate oxidase mutants. A: SDS-PAGE identification. M: medium molecular weight pre-stained protein marker; 1: R291K; 2: E208D; 3: R249Q. B: HPLC identification.

Figure 4 is a comparison of the enzymatic activities of highly active mammalian uricase mutants and wild-type canine uricase.

Detailed ways

In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments. Examples of such embodiments are shown in the drawings. It should be understood that the specific examples described in the following embodiments of the present invention are only used to illustrate the specific embodiments of the present invention and are intended to explain the present invention, but do not constitute a limitation of the present invention.

The endpoints of ranges and any values disclosed herein are not limited to the precise range or value, but such ranges or values should be understood to include approximations of these ranges.

The present invention provides a highly active mammalian urate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1.

Through systematic research, the inventor found that as evolution proceeds, the activity of urate oxidase in mammals of various species tends to become lower and lower. Through multiple sequence alignment of mammalian uricases with high homology to human uricases, the selection of highly conserved or shared amino acid sequences may rule out the accumulation of mammalian uricases in a single species during the evolution process. Missense mutations were used to obtain mammalian urate oxidase mutant proteins with higher activity.

Based on the protein sequences of mammalian urate oxidases such as dogs, pigs, cattle, rabbits, rats, and nocturnal monkeys, the above wild-type mammalian urate oxidases have been confirmed by the inventor to have the activity of degrading uric acid, and the urate oxidase protein sequences are complete It is 300-304 amino acids long; analyzed using software such as Blast, the identity (Identity) between the wild-type mammalian urate oxidase and the deduced human urate oxidase sequence should be no less than 85.0%. Based on the amino acid sequences of more than 6 kinds of mammalian uricases mentioned above, through multi-species sequence alignment (Alignment), the highly conserved regions are retained, and one or more alternative amino acids are designed through consensus analysis in the non-conserved variable regions. constitute.

More specifically, when there are large differences in the amino acid sequences of the variable regions between multiple species, the 1-2 amino acids with the highest conservation or commonality are first selected, such as at position 52, dog, pig, rabbit, nocturnal monkey, and artificial serine , cow is asparagine, rat is arginine, serine is significantly more common than the other two amino acids, so serine with the highest commonality is selected at position 52; for example, at position 291, canine source, cattle source, porcine source The source is arginine, rabbit, rat, night monkey, artificial lysine, there is no obvious difference in the commonality of arginine and lysine, then arginine and lysine are selected respectively at position 291 to construct Two alternative mutant sequences. By analogy, the most common amino acid or amino acids are selected at amino acid positions with different sequences, and a mammalian uricase mutant library is constructed. The amino acid sequence of the MU protein with the highest conservation or consensus among multiple species of mammalian uricases is obtained, which is a highly active mammalian uricase mutant as shown in SEQ ID NO: 1.

In order to further improve the activity and thermal stability, the present invention also uses methods including but not limited to the use of whole gene synthesis to synthesize the DNA sequence corresponding to the above amino acid sequence for the mutant shown in SEQ ID NO: 1, and use site-directed mutagenesis. , constructing different mammalian uricase mutant DNA sequences. The method of site-directed mutation can be obtained by various techniques such as DNA recombination and PCR that are well known to those skilled in the art, including but not limited to the two-round staggered extension PCR method and the site-directed mutation method described in Strantagene's quick change.

Further, on the basis of what is shown in SEQ ID NO: 1, the mutations E208D and R249Q can improve the enzyme specific activity and thermal stability. Therefore, the present invention also provides a highly active mammalian urate oxidase mutant. The mutations The body contains mutations E208D and/or R249Q compared to SEQ ID NO: 1. The E208D refers to the mutation of glutamic acid at position 208 of SEQ ID NO: 1 to aspartic acid; R249Q refers to the mutation of arginine at position 249 of SEQ ID NO: 1 to glutamine.

In some embodiments, the mutant may also include other suitable mutations, as long as the mutations do not significantly reduce the enzyme specific activity. In some embodiments, the mutant has an enzyme specific activity increased by at least 25% compared to wild-type canine urate oxidase as shown in SEQ ID NO: 3.

In some embodiments, the amino acid sequence of the mutant is as shown in any one of SEQ ID NO: 1, 5 or 7, in which case the sequence of the mutant is as shown in SEQ ID NO: 1, or contains the single mutation E208D or R249Q .

According to the highly active mammalian uricase mutant, the amino acid sequence can be synthesized into the corresponding DNA sequence or mRNA sequence for producing the uricase mutant. The present invention also provides a nucleic acid molecule comprising a sequence encoding a highly active mammalian uricase mutant according to any aspect of the present invention; preferably, the nucleic acid molecule is DNA or RNA. When the nucleic acid molecule is DNA, the urate oxidase mutant is synthesized through DNA translation and expression for protein production; the RNA can be mRNA for transient expression in cells.

The present invention also provides a gene expression cassette, which contains a promoter and a coding sequence operably connected behind the promoter, and the coding sequence encodes a protein according to any aspect of the present invention. Sequence of highly active mammalian urate oxidase mutants.

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

Through the correspondence between amino acids and nucleotides and codon optimization, those skilled in the art can obtain different nucleotide sequences. In some embodiments, the nucleotide sequence of the coding sequence is as set forth in any of SEQ ID NO: 2, 4, or 6.

The present invention also provides an expression vector, which includes the gene expression cassette described in any aspect of the present invention. The expression vector is preferably a plasmid vector, which can be expressed in mammalian cells, insects, yeast, bacteria or other cells through cell transduction.

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

In some embodiments, the host cells are selected from mammalian cells, insect, yeast or bacterial cells; preferably Enterobacteriaceae cells or yeast cells.

The present invention also provides a method for producing a highly active mammalian urate oxidase mutant according to any aspect of the present invention, including: expressing the mutant in the host cell, and isolating and purifying the mutant.

The mammalian uricase mutant protein of the present invention can be isolated from inside or outside the host cell (such as culture medium) and purified into a high-purity homogeneous protein. This method of protein separation and purification is not limited to any specific 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, reversed phase chromatography, etc. can be applied. These chromatographies can be performed with liquid chromatography such as fast protein liquid chromatography systems. Use common protein detection methods to detect protein purity and concentration, such as HPLC method, SDS-polyacrylamide electrophoresis method, isoelectric point electrophoresis method, BCA method, Lowry method, Kjeldahl method, etc. Use the uric acid substrate degradation method to detect the specific activity of urate oxidase. For example, based on the characteristic absorption peak of the substrate uric acid at 293 nm, use a UV spectrophotometer or high-performance liquid chromatography method to detect the uric acid consumption rate, and calculate the urate oxidase activity per unit volume. The specific activity of urate oxidase protein was calculated based on the monomer volume urate oxidase activity and unit volume urate oxidase protein concentration. Detect the thermal stability of urate oxidase through methods such as 25-37°C thermal stability testing. Even better, detect the thermal stability of urate oxidase through methods such as 37°C thermal stability testing.

The invention also provides the use of the highly active mammalian urate oxidase mutant in the preparation of urate oxidase drugs. The urate oxidase drug can be used to treat hyperuricemia and gout.

Example 1 Bioinformatics analysis and initial mutant design of multi-species mammalian urate oxidase

Based on the sequence of the human urate oxidase pseudogene (GenBank: AB074326.2), which was translated into an amino acid sequence, the nonsense mutations at position 33 and position 187 were replaced with arginine and arginine of the night monkey-derived urate oxidase, respectively. acid. The protein sequence was subjected to Blast analysis to obtain a mammalian urate oxidase amino acid sequence with an identity of no less than 85%. The above-mentioned mammalian urate oxidase amino acid sequence was subjected to multi-sequence alignment analysis using Clustal Omega and other software (as shown in Figure 1). The most conserved or shared MU protein amino acid sequence of multi-species mammalian urate oxidase was obtained as follows:

MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVD FEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL (SEQ ID NO: 1)

Based on the above amino acid sequence, DNA was designed according to the preferred codons of E. coli and yeast, and the corresponding company was entrusted to perform full gene synthesis. The corresponding DNA sequence is:

CATATGGCCCATTATCATAATGATTATAAAAAAAATGATGAAGTTGAATTTGTTCGTACCGGTTATGGTAAAGATATGGTTAAAGTTCTGCATATTCAGCGTGATGGTAAATATCATTCTATTAAAGAAGTTGCCACCTCTGTTCAGCTGACCCTGTCTTCTAAAAAAGATTATCTGCATGGTGATAATTCTGATATTATTCCAACCGATACCATTAAAAATACCGTTCATGTTCTGGCCAAATTAAAGGTATTAAATCTATTGAAACC TTTGCCATGAATATTTGTGAACATTTTCTGTCTTCTTTTAATCATGTTATTCGTGCCCAGGTTTATGTTGAAGAAGTTCCATGGAAACGTTTTGAAAAAAATGGTGTTAAACATGTTCATGCCTTTATTCATACCCCAACCGGTACCCATTTTTGTGAAGTTGAACAGCTGCGTTCTGGTCCACCAGTTATTCATTCTGGTATTAAAGATCTGAAAGTTCTGAAAACCACCCAGTCTGGTTTTGAAGGTTTTTACCACCGATCAGTTTTACCACC CTGCCAGAAGTTAAAGATCGTTGTTTTGCCACCCAGGTTTATTGTAAATGGCGTTATCATCAGGGTCGTGATGTTGATTTTGAAGCCACCTGGGATACCGTTCGTGATATTGTTCTGGAAAAATTTGCCGGTCCTTATGATAAAGGTGAATATTCTCCATCTGTTCAGAAAACCCTGTATGATATTCAGGTTCTGTCTCTGTCTCGTGTTCCAGAAATTGAAGATATGGAAATTTCTCTGCCAAATTTTCATTATTATTATTATTG ATATGTCTAAAATGGGTCTGATTAATAAAGAAGAAGTTCTGCTGCCACTGGATAATCCTTATGGTAAAATTACCGGTACCGTTAAACGTAAACTGAGCTCTCGTCTGTGATAAGGATCC (SEQ ID NO: 2).

After amplifying the recombinant plasmid synthesized by the whole gene, double digest it with NdeI and BamHI, recover the target fragment, use T4 DNA ligase to connect it to the plasmid pET-3C (Invitrogen) that has also been digested with NdeI and BamHI, and use "Current" The ligation mixture was transferred into the E. coli cloning host strain TOP10 according to the standard method described in "Protocols in Molecular Biology".

Transformation reaction is carried out on LB plates containing ampicillin. After growing the transformants overnight, single clone colonies after transformation are picked to prepare plasmids. The recombinant plasmid pET-3C-MU is screened by enzyme digestion and PCR verification, and is confirmed after DNA sequencing. The MU sequence in the positive recombinant plasmid was completely consistent with the theoretical sequence.

Example 2 Recombinant expression of urate oxidase in multiple species of mammals

The above-mentioned correctly sequenced MU recombinant plasmid was transformed into E. coli expression host bacteria and expressed. Use E. coli BL21(DE3), BL21 Star(DE3) or BL21 Star(DE3)plysS to express MU protein. These strains are just some of the many suitable for expressing chimeric proteins and are commercially available from Novagen, Invitrogen and Stratagen respectively. Transformants were identified by their ability to grow on LB plates containing ampicillin.

The Escherichia coli expression recombinant strain containing the MU recombinant plasmid was cultured overnight in a liquid LB medium containing 50ug/ml ampicillin, and the above culture liquid was inoculated into a large culture at an inoculation ratio of 1:100. After these cells grow to a certain optical density at 600 nm, add IPTG to a final concentration of 0.5mM to induce expression of the target protein, and continue to culture the cells for 3 hours. The cells were then harvested by centrifugation, and the pellet was washed with 50mM Tris buffer, centrifuged and stored at -20 degrees, and subjected to SDS-PAGE detection. The expressed protein band was around 35kDa (as shown in Figure 2).

Example 3 Design of multispecies mammalian urate oxidase mutant library

Using the MU protein DNA sequence as the original template, mutation sites are designed respectively according to the level of commonality, for example: Q109H (mutation is represented by a triplet: letter-number-letter, where the number indicates the position of the mutated amino acid, and the letter before the number Corresponding to the amino acid designed for mutation, the letter after the number indicates the amino acid used to replace the amino acid before the number. The numbers are ordered according to the 304th amino acid of human uricase), L146M, S148N, E208D, T213A, S246T, R249Q, V250L.

Specifically, a single point mutation was carried out based on MU to construct a mutant library, and recombinant expression, preparation and enzyme specific activity detection were carried out according to the methods of Examples 3, 4 and 5 to screen out beneficial mutations that improve the enzyme specific activity and eliminate harmful ones. Mutation, screening mammalian uricase mutants with optimal enzyme specific activity. The inventor found through systematic research in the early stage that the wild-type canine uricase protease protein (SEQ ID NO: 3) has higher specific activity, so the uricase mutant of the present invention and the recombinant wild-type canine uricase protein (named wCU) to conduct comparative studies. The amino acid sequence of wild-type canine uricase is as follows:

MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSV

QLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSF

NHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIH

SGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVD

FEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEIS

LPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTAKRKLASKL (SEQ ID NO: 3).

DNA containing the target mutation was prepared using staggered extension PCR mutagenesis. The following is an example of preparing Q109H:

Primer number DNA sequence Primer 1 (SEQ ID NO: 8) 5'CACGACATATGGCCATTATCATA3' Primer 2 (SEQ ID NO: 9) 5'GGATCCTTATCACAGACGAGAGCT3' Primer 3 (SEQ ID NO: 10) 5'CGTGCCCACGTTTATGTTGAAGAA3' Primer 4 (SEQ ID NO: 11) 5'ATAAACGTGGGCACGAATAACATG3'

Preparation of MU _Q109H : First-stage PCR: The template sequence is the whole gene synthesis sequence in Example 1 (SEQ ID NO: 2), and the primers are primer 1 and primer 3 in the table. The PCR reaction system and method all use PCR reaction kits, as follows Merchant's instructions settings. PCR reaction conditions were: 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min, a total of 30 cycles. The first cycle was denaturation at 94°C for 10 min, and the last cycle was extension at 72°C for 10 min. Amplify the product MU _Q109H -a fragment according to the above PCR conditions; second stage PCR: the template sequence is the same as the first stage PCR, and the primers are primer 2 and primer 4. Amplify according to the above PCR conditions to obtain the MU _Q109H -b fragment; third stage PCR Stage PCR: The template is a 1:1 mixed solution of MU _Q109H -a fragment and MU _Q109H -b fragment, and the primers are primer 1 and primer 2. The product MU _Q109H is amplified according to the above PCR conditions. The DNA sequence containing the mutated sequence was double digested with NdeI and BamHI. The ligation, transformation, screening and expression methods were the same as in Example 2.

Example 4 Expression and purification of mammalian uricase mutants

Take 50g of bacterial sediment and add 500ml of sterilization solution. The pH of the sterilization solution is 8.3, containing 25mM Tris-HCl, 5mMEDTA, and 0.1mg/ml lysozyme. Stir at 37°C for 60 to 80 minutes, then add 1mM MgCl ₂ ·6H ₂ O. Stir 1 μg·mL ^-1 nuclease overnight; centrifuge at 4°C, 8500r·min ^-1 for 20 min to collect the bacterial precipitate. Take the broken bacteria precipitate and suspend the precipitate in 1L washing liquid (25mM Tris-HCl, 5mM EDTA, 1.5% TritonX-100, pH 8.3) according to 100g of wet bacteria. After evenly dispersed, stir at 30°C for 2h; 8500r·min ^-1 , 4 Centrifuge at 15°C for 15 minutes, wash the precipitate with the same washing solution as above, and collect the precipitate by centrifugation. Take the TritonX-100 washed pellet and suspend the pellet in 1L washing solution (PBS, 0.34μg·mL ^-1 nuclease) for 100g of wet bacteria. After even dispersion, stir at 30℃ for 2h, centrifuge at 8500r·min ^-1 and 4℃ for 15min. Collect the precipitate by centrifugation. Wash the precipitate with PBS and nuclease, and suspend the precipitate in 1 liter of washing liquid (PBS) for 100 grams of wet bacteria. After homogenization, stir at 30°C for 2 hours, centrifuge at 8500r·min ^-1 and 4°C for 15min, and take the precipitate. Wash once with the same washing solution as above, and centrifuge to collect the precipitate. Take the precipitate after sterilization and washing, and dissolve 10g of precipitate in 3L dissolution buffer (0.1M Na ₂ CO ₃ -NaHCO ₃ , pH 10.3). After homogenization with a homogenizer, stir at room temperature overnight. Centrifuge at 8500r·min ^-1 and 4℃ for 15min, and collect the centrifugation supernatant. Add (NH ₄ ) ₂ SO ₄ to the supernatant with a final concentration of 10% saturation, let it stand at 4°C overnight, centrifuge at 8500r·min ^-1 for 15min at 4°C, and collect the centrifugal precipitate; dissolve in the dissolving buffer and stir at room temperature overnight. , 8500r·min ^-1 , centrifuge for 15min at 4°C, and collect the centrifugation supernatant. The supernatant is purified on a DEAE agarose anion exchange chromatography column (GE). After all the target proteins are hung on the column, a linear gradient of 0 to 0.2M NaCl (pH 10.3, 0.1M Na ₂ CO ₃ -NaHCO ₃ ) is used. Elute, the target protein is eluted at 0.1M NaCl; the above-mentioned elution component DEAE agarose anion exchange chromatography column (GE) is concentrated, and it is washed with 0.2M NaCl (H is 10.3, 0.1M Na ₂ CO ₃ -NaHCO ₃ ) eluent elutes the target protein. At this time, SDS-PAGE and HPLC were used to detect the purity, both of which could reach over 95% (as shown in Figure 3).

Example 5 Activity detection of mammalian uricase mutant protein

At 37°C and pH 8.6, the amount of enzyme that converts 1 μmol of uric acid into allantoin per minute is defined as one international unit (IU). Uric acid has a characteristic absorption peak at 293nm. When it is degraded by uricase, the product has no absorption peak in this wavelength range. The change in absorbance at 293nm is regularly detected to determine the reduction of uric acid, and then the molar extinction coefficient of uric acid (1.23 ×104M ^-1 ·CM ^-1 ) to calculate the uric acid concentration, and the uricase activity can be calculated based on changes in the uric acid concentration. Adjust the UV spectrophotometer to 293nm. After the machine is stable, use 0.1M sodium tetraborate solution as a blank to zero, take 3ml of 0.1mM uric acid solution, react and dissolve it, add it to a quartz cuvette, and add 10ul of uricase mutant protein. Take a reading every 30 seconds and measure the change in OD293 within 2 minutes. According to the formula C=A/Kb (C is the concentration of uric acid in the solution, A is the absorbance value at 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 concentration; calculate the amount of reduced uric acid material according to △M=△CV (△M is the number of moles of uric acid reduced, △C is the change in uric acid concentration, and C is the volume of the reaction solution); according to U=△M/TV1 (U is the unit of uricase activity per milliliter of plasma, T is the reaction minutes, and V1 is the volume of uricase mutant protein added to the reaction system) Calculate the uricase activity. Among them, the specific activities of MU, MU _E208D (SEQ ID NO: 5), and MU _R249Q (SEQ ID NO: 7) are approximately 29.8%, 28.9%, and 34.3% higher than wild-type canine uricase respectively (as shown in Figure 4 ).

Example 6 Detection of thermal stability of mammalian uricase mutants

Dilute the uricase mutant protein to 1mg/ml with buffer (0.1M Na ₂ CO ₃ -NaHCO ₃ , pH 10.3), place it in a 37°C incubator, and take out some samples after 0h, 0.5h, and 1h. Determine uricase activity according to the method in Example 5, and compare the enzyme activity retention rate.

The results showed that the thermal stability of the three uricase mutants was better than that of wild-type canine uricase, and MU _E208D had the best thermal stability.

Other sequences involved in the embodiments

MU _E208D nucleotide sequence:

CATATGGCCCATTATCATAATGATTATAAAAAAAATGATGAAGTTGAATTTGTTCGTACCGGTTATGGTAAAGATATGGTTAAAGTTCTGCATATTCAGCGTGATGGTAAATATCATTCTATTAAAGAAGTTGCCACCTCTGTTCAGCTGACCCTGTCTTCTAAAAAAGATTATCTGCATGGTGATAATTCTGATATTATTCCAACCGATACCATTAAAAATACCGTTCATGTTCTGGCCAAATTAAAGGTATTAAATCTATTGAAACC TTTGCCATGAATATTTGTGAACATTTTCTGTCTTCTTTTAATCATGTTATTCGTGCCCAGGTTTATGTTGAAGAAGTTCCATGGAAACGTTTTGAAAAAAATGGTGTTAAACATGTTCATGCCTTTATTCATACCCCAACCGGTACCCATTTTTGTGAAGTTGAACAGCTGCGTTCTGGTCCACCAGTTATTCATTCTGGTATTAAAGATCTGAAAGTTCTGAAAACCACCCAGTCTGGTTTTGAAGGTTTTTACCACCGATCAGTTTTACCACC CTGCCAGAAGTTAAAGATCGTTGTTTTGCCACCCAGGTTTATTGTAAATGGCGTTATCATCAGGGTCGTGATGTTGATTTTGATCCACCTGGGATACCGTTCGTTGATATTGTTCTGGAAAAATTTGCCGGTCCTTATGATAAAGGTGAATATTCTCCATCTGTTCAGAAAACCCTGTATGATATTCAGGTTCTGTCTCTGTCTCGTGTTCCAGAAATTGAAGATATGGAAATTTCTCTGCCAAATTCATTATTATTTTAATATTG ATATGTCTAAAATGGGTCTGATTAATAAAGAAGAAGTTCTGCTGCCACTGGATAATCCTTATGGTAAAATTACCGGTACCGTTAAACGTAAACTGAGCTCTCGTCTGTGATAAGGATCC (SEQ ID NO: 4)

Corresponding amino acid sequence:

MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVD FDATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO: 5)

MU _R249Q nucleotide sequence:

CATATGGCCCATTATCATAATGATTATAAAAAAAATGATGAAGTTGAATTTGTTCGTACCGGTTATGGTAAAGATATGGTTAAAGTTCTGCATATTCAGCGTGATGGTAAATATCATTCTATTAAAGAAGTTGCCACCTCTGTTCAGCTGACCCTGTCTTCTAAAAAAGATTATCTGCATGGTGATAATTCTGATATTATTCCAACCGATACCATTAAAAATACCGTTCATGTTCTGGCCAAATTAAAGGTATTAAATCTATTGAAACC TTTGCCATGAATATTTGTGAACATTTTCTGTCTTCTTTTAATCATGTTATTCGTGCCCAGGTTTATGTTGAAGAAGTTCCATGGAAACGTTTTGAAAAAAATGGTGTTAAACATGTTCATGCCTTTATTCATACCCCAACCGGTACCCATTTTTGTGAAGTTGAACAGCTGCGTTCTGGTCCACCAGTTATTCATTCTGGTATTAAAGATCTGAAAGTTCTGAAAACCACCCAGTCTGGTTTTGAAGGTTTTTACCACCGATCAGTTTTACCACC CTGCCAGAAGTTAAAGATCGTTGTTTTGCCACCCAGGTTTATTGTAAATGGCGTTATCATCAGGGTCGTGATGTTGATTTTGAAGCCACCTGGGATACCGTTCGTGATATTGTTCTGGAAAAATTTGCCGGTCCTTATGATAAAGGTGAATATTCTCCATCTGTTCAGAAAACCCTGTATGATATTCAGGTTCTGTCTCTGTCTCAGGTTCCAGAAATTGAAGATATGGAAATTTCTCTGCCAAATTTTCATTATTATTATTATTG ATATGTCTAAAATGGGTCTGATTAATAAAGAAGAAGTTCTGCTGCCACTGGATAATCCTTATGGTAAAATTACCGGTACCGTTAAACGTAAACTGAGCTCTCGTCTGTGATAAGGATCC (SEQ ID NO: 6).

Corresponding amino acid sequence:

MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVD FEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSQVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL (SEQ ID NO: 7).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, many simple modifications can be made to the technical solution of the present invention, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the disclosed content of the present invention. All belong to the protection scope of the present invention.

Claims (10)

1. A high activity 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 comprises the mutations E208D and/or R249Q.

2. The mutant of claim 1, wherein the mutant hybridizes to a sequence set forth in SEQ ID NO:3, the specific enzyme activity is improved by at least 25% compared with the wild-type canine urate oxidase.

3. The mutant of claim 1, wherein the amino acid sequence of the mutant is as set forth in SEQ ID NO: 1.5 or 7.

4. A nucleic acid molecule comprising the coding sequence of the high activity 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. 4 or 6.

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 mutant highly active mammalian urate oxidase of 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 activity 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 active mammalian urate oxidase mutant according to any one of claims 1 to 3 for the preparation of a urate oxidase drug.