CN117587041A - Recombinant uricase, and preparation method and application thereof - Google Patents
Recombinant uricase, and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
- C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12N9/0048—Uricase (1.7.3.3)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
- C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
The application discloses recombinant uricase, and a preparation method and application thereof. The application provides a polynucleotide sequence for encoding uricase, which is optimized by synonymous codon preference, and has high expression efficiency in escherichia coli; the vector adapting to the polynucleotide sequence is also provided, and the high-activity uricase with specific activity as high as 32.2U/mg is prepared and obtained through the cooperative coordination of the vector and the polynucleotide sequence; in addition, the method for preparing the recombinant uricase is provided, the method not only improves the yield of the uricase, but also ensures that the obtained product has extremely high content of soluble protein, is easy to purify, simplifies the post-treatment step and saves the production cost.
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to recombinant uricase, a preparation method and application thereof.
Background
Uricase is an enzyme that catalyzes the reaction of uric acid hydrolysis to allantoin, hydrogen peroxide, and carbon dioxide, and may be used to determine uric acid in blood or urine. Uricase is composed of four identical subunits, each with a molecular weight of about 34kD and composed of 301-304 amino acids, and four uricase monomers are non-covalently bound into a homotetramer, wherein first two monomer proteins are connected end to form a cyclic dimer; then, two cyclic dimers are overlapped up and down to form a tetramer, uricase structures, optimal enzyme activity pH and isoelectric points of uricase from different species are different, uricase from animal sources is a tetramer, and each enzyme molecule contains four copper atoms; uricase derived from Candida utilis does not contain metal ions such as Cu 2+ And Fe (Fe) 3+ Nor contains coenzyme, and cysteine residue in the molecule does not participate in enzymatic activity reaction; uricase produced by Bacillus fastidious and Huang Qumei also does not contain metal ions and coenzymes.
Uricase is currently mainly applied to three aspects of diagnostic reagents, clinical therapeutic drugs, biosensors and the like, for example, uricase is used for catalyzing uric acid oxidative discoloration to determine uric acid content so as to diagnose gout and hyperuricemia. The uricase is prepared by a common extraction method from fermentation liquids such as aspergillus niger, aspergillus flavus and the like, but the downstream separation and purification are complex due to long fungus fermentation period, the production cost is high, and the large-scale production is difficult. The uricase production method based on bioengineering becomes a more efficient production mode. In the prior art, patent CN104342415A discloses a preparation method of recombinant uricase, and the method successfully expresses uricase in escherichia coli, but the inventor discovers that the protein yield of the method is still low in research, and the activity of the prepared product is insufficient. Therefore, there is still a need to develop a method for efficiently producing highly active uricase.
Disclosure of Invention
The invention aims to provide a preparation method of recombinant uricase with high yield and good product activity.
It is another object of the present invention to provide a polynucleotide sequence encoding uricase.
It is another object of the present invention to provide a vector that is adapted to a polynucleotide sequence encoding uricase.
It is another object of the present invention to provide a kit comprising a polynucleotide sequence encoding uricase.
To solve the above technical problem, according to a first aspect of the present invention, there is provided a polynucleotide encoding uricase, the polynucleotide being codon optimized, and the polynucleotide being selected from any one of the following:
(i) A polynucleotide having a sequence as shown in SEQ ID NO. 1;
(ii) A polynucleotide having a homology of greater than 95% with the sequence shown in SEQ ID NO. 1; and
(iii) Having a polynucleotide complementary to the polynucleotide sequence set forth in (i) or (ii).
In some preferred embodiments, the polynucleotide encoding uricase is obtained from the polynucleotide sequence encoding uricase in Arthrobacter sphaeroides via codon optimization.
In a second aspect of the invention there is provided an expression vector comprising a polynucleotide provided in the first aspect of the invention.
In some preferred embodiments, the expression vector comprises a polynucleotide sequence that expresses a His X6 tag, more preferably, the expression vector has the 3' end of the polynucleotide linked to a polynucleotide sequence that expresses a His X6 tag.
In some preferred embodiments, the expression vector is an E.coli expression vector, more preferably pS28a (+).
In a third aspect of the invention there is provided a host cell comprising an expression vector provided in the second aspect of the invention; or alternatively
The host cell has integrated into its genome a polynucleotide as provided in the first aspect of the invention.
In some preferred embodiments, the host cell is E.coli (Escherichia coli).
In some preferred embodiments, the host cell is an E.coli BL21 (DE 3) strain.
In a fourth aspect, the present invention provides a method of preparing uricase, the method comprising the steps of: culturing the host cell of the third aspect of the invention to express the protein of interest; and
separating the target protein to obtain the uricase;
wherein the target protein has an amino acid sequence shown as SEQ ID NO. 2.
In some preferred embodiments, the host cell is obtained by transforming E.coli with a plasmid comprising a polynucleotide according to the first aspect of the invention.
In some preferred embodiments, the host cells are cultured in TB medium.
In some preferred embodiments, the host cell is cultured in a shaking environment.
In some preferred embodiments, the medium used in culturing the host cell comprises a kanamycin resistance gene.
In some preferred embodiments, the host cell is cultured using IPTG to induce expression of the protein of interest.
In some preferred embodiments, the host cell is cultured until an OD600 of 0.6 to 0.8 is reached, and is then induced with IPTG to express the protein of interest.
In some preferred embodiments, the step of isolating the protein of interest comprises:
passing the crushed target protein supernatant through a chromatographic column, eluting, and collecting the eluent.
In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column, such as a HisTrapTMFF.
In a fifth aspect, the invention provides a kit comprising: a polynucleotide as provided in the first aspect of the invention; or alternatively
An expression vector as provided in the second aspect of the invention; or alternatively
A host cell according to the third aspect of the invention; or alternatively
Or uricase prepared according to the method of the fourth aspect of the invention.
Compared with the prior art, the invention has at least the following advantages:
(1) The invention provides a polynucleotide sequence for coding uricase, which is optimized by synonymous codon preference, and has high expression efficiency in escherichia coli;
(2) The invention also provides a vector adapting to the polynucleotide sequence, and the high-activity uricase is prepared and obtained through the cooperative coordination of the vector and the polynucleotide sequence, and the specific activity is as high as 32.19U/mg;
(3) The invention provides a method for preparing recombinant uricase, which not only improves the yield of uricase, but also has the advantages of extremely high content of soluble protein of the obtained product, easy purification and simplified post-treatment steps.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.
FIG. 1 is a diagram showing the SDS-PAGE identification of uricase according to an embodiment of the present invention;
FIG. 2 is a diagram of uricase via HisTrap in accordance with an embodiment of the invention TM Electrophoresis after FF affinity purification;
FIG. 3 is a graph of absorbance versus uricase concentration for an example according to the invention.
Detailed Description
The inventor provides an expression system for preparing uricase by efficiently expressing heterologous genes in microorganisms through extensive and intensive research, and the polynucleotide sequences matched with a target escherichia coli vector are obtained through codon preference optimization in the expression system, so that the yield of uricase is greatly improved, the subsequent purification steps are simplified, the economic cost is saved, and the activity of recombinant uricase obtained by a uricase preparation method based on the expression system is greatly improved, thereby having good application prospect. The embodiment of the invention constructs a method for efficiently preparing high-activity uricase, which comprises the following steps: obtaining a target gene sequence; carrying out synonymous codon preference optimization on a target gene sequence; obtaining a vector of a target gene, and connecting the target gene to the vector; transforming a host cell with a vector comprising a gene of interest; culturing the host cell to express the target protein uricase; and separating the target protein.
[ obtaining a Gene of interest/obtaining a nucleic acid sequence related to a protein of interest ]
The full-length nucleotide sequence of the target protein or its element or a fragment thereof of the present invention can be usually obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the genes of the present invention. Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
In one embodiment of the present invention, the amino acid sequence of the target protein (SEQ ID NO: 2) is analyzed by NCBI database to obtain the sequence information of the target gene. The amino acid sequence of Arthrobacter sphaeroides uricase is analyzed, for example, by NCBI database, to obtain the gene sequence of interest encoding the same.
Synonymous codon preference optimization
To overcome the potential problem of reduced yield when expressing heterologous proteins in E.coli, the present invention relates to synonymous codon-biased optimized polynucleotide sequences. The obtained target gene sequence is subjected to synonymous codon preference optimization, the target gene sequence (SEQ ID NO: 1) subjected to synonymous codon preference optimization can express the same amino acid sequence as the target protein, but the stability and efficiency of the expression process are improved, and finally the obtained target protein maintains higher activity.
The invention also relates to polynucleotides having a homology of more than 95% with the sequence shown in SEQ ID NO. 1; and a polynucleotide complementary to the sequence shown in SEQ ID No. 1.
In order to improve the solubility of the obtained product and reduce inclusion bodies, the target gene sequence is obtained by extracting uricase from Arthrobacter sphaeroides as a source and performing reverse transcription.
[ vector for target Gene ]
The invention also relates to vectors comprising the polynucleotides of the invention. "vector" in the present invention means a linear or circular DNA molecule comprising a fragment encoding a protein of interest operably linked to other fragments providing for its transcription. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, a vector, and the like. The vector fragment may be derived from the host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, synthetic or conveniently subjected to recombinant DNA procedures, the choice of vector generally being dependent on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one that, when introduced into the host cell, integrates into the host cell genome and replicates with the chromosome with which it is integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the present invention, pS28a (+) was selected as a vector to obtain higher expression efficiency.
Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequences of the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Illustratively, the insertion of the exogenous DNA fragment is accomplished by cleaving the vector DNA molecule with a DNA endonuclease into a linear molecule that can be linked to the exogenous gene, and then ligating the codon optimized fragment of the gene of interest to the vector, optionally with a single restriction site cohesive end ligation, double restriction site directional cloning, cohesive end ligation of different restriction sites, blunt end ligation, artificial linker ligation, or end ligation with an oligonucleotide.
In one embodiment of the invention, the vector further comprises a His X6 tag expressing polynucleotide sequence, preferably the His X6 tag expressing polynucleotide sequence is ligated to the 3' end (C-terminus) of the gene sequence of interest.
[ vector-transformed host cell containing the Gene of interest ]
The invention also relates to host cells genetically engineered with the vector or fusion protein coding sequences of the invention. The vector containing the codon-optimized gene of interest may be inserted, transfected or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest. A "host cell" in the present invention is a cell into which an exogenous polynucleotide and/or vector has been introduced. The host cell may be a eukaryotic host cell or a prokaryotic host cell, the host cell is preferably a bacterium, and is preferably E.coli, more preferably E.coli BL21 (DE 3) strain (Escherichia coli BL (DE 3) strain).
[ method for producing target protein ]
The invention also relates to a method for preparing the target protein, and the polynucleotide sequence can be used for expressing or producing recombinant protein. Generally, there are the following steps:
(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;
(2) A host cell cultured in a suitable medium;
(3) Separating and purifying the protein from the culture medium or the cells.
Wherein, the transformation or transduction of the recombinant expression vector containing the polynucleotide of the step (1) into a suitable host cell can be performed by conventional techniques well known to those skilled in the art, and when the host is E.coli, a heat shock method, an electrotransformation method, etc. can be selected.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time. In order to promote expression of the target protein, a preferred embodiment of the present invention uses a host cell cultured in a TB medium, and the medium used contains a kanamycin resistance gene.
The protein in the above method may be expressed in the cell, or on the cell membrane, or secreted outside the cell. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. In one embodiment of the invention, affinity chromatography is used to molecular target proteins.
In the present disclosure, any exemplary or exemplary language (e.g., ") provided for certain embodiments herein is used merely to better present the disclosure and does not limit the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
If the definition or use of a term in a reference is inconsistent or inconsistent with the definition of that term described herein, the definition of the term described herein applies and the definition of the term in the reference does not apply.
Various terms used herein are shown below. If a term used in a claim is not defined below, the broadest definition persons in the pertinent art have given that term are given as reflected in publications or issued patents that are printed at the time of application.
As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., a nucleic acid or polypeptide) that the nucleic acid or polypeptide is found in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in solvents, buffers, ions or other components that are normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.
As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
The invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.
As used herein, the term "codon optimization" refers to a manner of improving the efficiency of gene synthesis by avoiding the use of low-availability or rare codons according to codon usage differences exhibited by organisms (including e.coli, yeast, mammalian blood cells, plant cells, insect cells, etc.) that actually do protein expression or production.
As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. The nucleotide or amino acid sequence of a polynucleotide or polypeptide is aligned, the number of positions in the aligned polynucleotide or polypeptide that contain the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotide or polypeptide that contain a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, according to the inclusion of different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertions or deletions of one or two nucleotides in the polynucleotide). The polypeptides may differ at one position, for example, by containing an amino acid (i.e., substitution or variation) or a deletion of an amino acid (i.e., an amino acid or deletion of an amino acid inserted into one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.
As used herein, the terms "sequence complementary" and "reverse sequence complementary" are used interchangeably to refer to a sequence that is opposite in direction to and complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is actaac, then its reverse complement is GTTCAT.
As used herein, the term "expression" includes any step involving the production of a polypeptide in a host cell, including, but not limited to, transcription, translation, post-translational modification, and secretion. After expression, the host cells or expression products can be harvested, i.e.recovered.
The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, it is to be noted that the terms used herein are used merely to describe specific embodiments and are not intended to limit the exemplary embodiments of this application.
Example 1 construction of uricase plasmid
(1) Uricase from different strains was selected and its amino acid sequence information was obtained as shown in Table 1. The gene sequences are obtained after amino acid sequence analysis, synonymous codon preference optimization is carried out on the gene sequences, and the connecting carrier is pET28a (+), and the C-terminal fusion expression (His) 6 tag.
(2) Recombinant plasmid transformed E.coli BL21 (DE 3)
Taking 1 mu L of plasmid, adding the plasmid into 30 mu L of escherichia coli competent BL21 (DE 3) under ice bath condition, standing for 20min in ice bath, carrying out heat shock for 45s in water bath at 42 ℃, standing for 2min on ice immediately, adding 400 mu L of SOC culture medium without antibiotics, and carrying out shaking culture at 220rpm for 50min at 37 ℃. mu.L of the bacterial liquid was uniformly spread on LB plates containing 100. Mu.g/mL of kana resistance, and incubated overnight at 37 ℃.
TABLE 1
| Bacterial origin | Amino acid sequence | Codon optimized nucleic acid sequences |
| Arthrobacter globosus | SEQ ID NO:2 | SEQ ID NO:1 |
| Pseudomonas aeruginosa | SEQ ID NO:4 | SEQ ID NO:3 |
| Candida | SEQ ID NO:6 | SEQ ID NO:5 |
SEQ ID NO:1：
GCTAGCATGAGCAACAAAATTGTGCTGGGCCATAATCAGTATGGCAAAGCGGAAGTGCGCGTGGTGAAAATTACCCGCGATACCGATCGCCATGAAATTGAAGATCTGAACGTGACGAGTCAGCTGCGCGGCGATTTTGAAGCGGCGCATCTGGAAGGCGATAACGCGCATGTGGTGGCGACCGATACGCAGAAAAACACCATTTATGCGTTTGCGCGCGAAGGCGTGGGCAGCCCGGAAGCGTTTCTGCTGCGCCTGGGCGAACATTTTACGAGCAGCTTTGATTGGGTGACCGGCGGCCGCTGGGAAGCGGAAAGCTATGCGTGGGAACGCATTCAAGCGCATGGCAGCGCGCATGATCATAGCTTTGTGCGCAAAGGCCAAGAAGTGCGCACCGCGGTGCTGGTGCGCGATGGCGCGGCGACCCATCTGATTAGCGGCCTGAAAGATCTGACCGTGCTGAAAAGCACGCAGAGCGGCTTTGTGGGCTATCCGAAAGATAAATATACCACCCTGCCGGAAACCACCGATCGCATTCTGGCGACCGATGTGAGCGCGCGCTGGCGCTTTCGCAGCGGCACCGATTTTAGCAGCCTGGATTTTAACAAAAGCTATGAAGATGTGAAAAGCCTGTTACTGGAAGGCTTTACCGAAAAATATAGCCATGCGCTGCAGCAGACCCTGTTTGATATGGGCGCGAAAGTGCTGGAAGCGCATAGCGAAATTGAAGAAATTAAATTTAGCATGCCGAACAAACATCATTTTCTGGTGGATCTGAGCCCGTTTGGCCTGGATAACCCGAACGAAGTGTTTTTTGCGGCGGATCGCCCGTATGGCCTGATTGAAGCGACCGTGCTGCGCGATGATGCGGAAGCGGCGGATGCGGCGTGGAGCGGCATTGCGGGCTTTTGCCATCACCATCACCATCATTAACTCGAG
SEQ ID NO:2：
MSNKIVLGHNQYGKAEVRVVKITRDTDRHEIEDLNVTSQLRGDFEAAHLEGDNAHVVATDTQKNTIYAFAREGVGSPEAFLLRLGEHFTSSFDWVTGGRWEAESYAWERIQAHGSAHDHSFVRKGQEVRTAVLVRDGAATHLISGLKDLTVLKSTQSGFVGYPKDKYTTLPETTDRILATDVSARWRFRSGTDFSSLDFNKSYEDVKSLLLEGFTEKYSHALQQTLFDMGAKVLEAHSEIEEIKFSMPNKHHFLVDLSPFGLDNPNEVFFAADRPYGLIEATVLRDDAEAADAAWSGIAGFCHHHHHH
SEQ ID NO:3：
ATGGCTGAATCTACTCAGCACAAACTGGACCGTGTTCGTCCACCGCGTGTGCAGATTACTTATGATGTCGAAATCGGCAACGCTATTGAGAAAAAAGAACTGCCGCTGGTTGTTGGTATTCTGGCTGATCTGTCCGGCAAACCGGACACTCCGCCTGCAAAGCTGGTAGAACGCCGCTTCGTTGATATCGACCGTGACAACTTCAACGAAATCCTGTCTTCCATCAGCCCGCGTGCTACCCTGCAGGTTGACAACACTATCTCCGGTGACGATTCTAAACTGAACGTTGAACTGCGTTTTAACCACATCGAAGATTTCGATCCAGTGAACCTGGTTAAACAGGTAGTTCCGCTGCGTCGTCTGTTTGAAGCTCGTCAGCGTCTGCGTGACCTGCTGACCAAACTGGATGGCAATGATGACCTGGATCAGCTGCTGCAGGATGTGGTTGCTAACACTGAAGGCCTGCAGGAAATCAAAGCGGCACGTCGTAAACCTAAACGTCCACGTCCAGCTACCGCTAACCCTCGTCCGACTTCTCGTCCGGACCCACTGCCTGGTGAAGCTGCAATGCCGAAGAGCAGCGCAGCGGAACAATCCGGCGAATCTTCCACCCAGACCCTGTCTCTGCTGGATGAAATCATCGCGAAGGGTCGTATGGCACACGACGATTCTCAGCAGGATTACGCTCGTGACATGCTGGCTGAATTTGCCACCCAGGTCCTGGACGAAGGTATGGCGGTTGATAAAGATACCGTTGCTATGATCAACGATCGTATTTCCCAGATTGATGCTCTGATTTCTGACCAGCTGAACCAGATCATCCACCACCCGGAGCTGCAGAAGCTGGAAGCAAGCTGGCGTGGTCTGCACCAGCTGGTTAGCAACACCGAAACCTCTGCCCGTCTGAAACTGCGTCTGCTGAATGTAGGTAAAAATGAGCTGCAGAACGACCTGGAGAAAGCAGTGGAGTTCGACCAGAGCGCACTGTTCAAAAAAATTTATGAAGAAGAATATGGTACCTTTGGCGGTCACCCGTTCTCTCTGCTGATCGGTGACTTCACCTTCGGTCGTCACCCGCAGGATATCGGTCTGCTGGAAAAACTGTCTAACGTTGCAGCAGCAGCACATGCTCCGTTCATCGCCGCTGCTTCTCCGCGTCTGTTCGACATGAACAGCTTCACCGAACTGGCCGTCCCGCGTGACCTGACTAAAATCTTCGAAAGCCTGGAACTGATTAAGTGGCGTGCTTTCCGTGAAAGCGAAGACAGCCGTTACGTATCTCTGGTACTGCCGAACTTCCTGCTGCGTCTGCCGTATGGTCCGGAAACTCGTCCGGTAGAAGGCATGAATTACGTCGAAGATGTGAATGGCACCGATCACAGCAAATACCTGTGGGGTAATGCGGCTTGGGTCCTGGCTCAGCGCATTACGGAGGCTTTCGCCAAGTATGGTTGGTGTGCAGCAATTCGTGGTGCAGAGGGTGGTGGCGCTGTTGAAGGTCTGCCGGCACATACTTTTCGTACCTCTTCTGGTGACCTGAGCCTGAAATGTCCGACCGAAGTGGCGATCACTGACCGTCGTGAAAAAGAACTGAACGACCTGGGCTTCATCTCCCTGTGTCACAAAAAGAACAGCGACGTCGCTGTTTTCTTTGGCGGCCAGACTACCAACAAAGCACGCCTGTACAACACCAATGAGGCAAACGCCAATGCGCGTCTGTCCGCGATGCTGCCATACGTTCTGGCAGCAAGCCGCTTCGCGCACTACCTGAAAGTTATCATGCGCGACAAAGTTGGCTCTTTCATGACGCGTGACAACGTACAGACCTATCTGAACAACTGGATCGCGGATTATGTGCTGATCAACGACAACGCGCCGCAGGAAATTAAAGCTCAGTACCCGCTGCGCGAAGCTCGTGTGGATGTTTCCGAAGTTGCAGGTAAGCCGGGTGCTTACCGTGCCACCGTTTTCCTGCGTCCGCACTTCCAGCTGGAAGAACTGAGCGCTTCCATTCGCCTGGTGGCGAATCTGCCTCCACCAGTTGCTGCT
SEQ ID NO:4：
MAESTQHKLDRVRPPRVQITYDVEIGNAIEKKELPLVVGILADLSGKPDTPPAKLVERRFVDIDRDNFNEILSSISPRATLQVDNTISGDDSKLNVELRFNHIEDFDPVNLVKQVVPLRRLFEARQRLRDLLTKLDGNDDLDQLLQDVVANTEGLQEIKAARRKPKRPRPATANPRPTSRPDPLPGEAAMPKSSAAEQSGESSTQTLSLLDEIIAKGRMAHDDSQQDYARDMLAEFATQVLDEGMAVDKDTVAMINDRISQIDALISDQLNQIIHHPELQKLEASWRGLHQLVSNTETSARLKLRLLNVGKNELQNDLEKAVEFDQSALFKKIYEEEYGTFGGHPFSLLIGDFTFGRHPQDIGLLEKLSNVAAAAHAPFIAAASPRLFDMNSFTELAVPRDLTKIFESLELIKWRAFRESEDSRYVSLVLPNFLLRLPYGPETRPVEGMNYVEDVNGTDHSKYLWGNAAWVLAQRITEAFAKYGWCAAIRGAEGGGAVEGLPAHTFRTSSGDLSLKCPTEVAITDRREKELNDLGFISLCHKKNSDVAVFFGGQTTNKARLYNTNEANANARLSAMLPYVLAASRFAHYLKVIMRDKVGSFMTRDNVQTYLNNWIADYVLINDNAPQEIKAQYPLREARVDVSEVAGKPGAYRATVFLRPHFQLEELSASIRLVANLPPPVAA
SEQ ID NO:5：
ATGCAGTCCGAACTGTACAGCTCTACCTACGGCAAAGCCAACGTTAAATTCCTGAAAGTCAAAAAAGATCAGTCTAACCCGACTGTACAGGAAATCCTGGAAGCTAACGTCCAGGTCCTGCTGCGTGGTAAATTCGAAGAGTCCTATACCAAAGCCGACAACTCTTCCATCGTTCCGACCGACACCGTTAAAAATACGATCCTGGTTGAAGCTAAAAACACCGACGTTTGGCCGATCGAACGTTTCGCAGCGCATCTGGCTAAACACTTTACTTCCAAATATGGTCACGTTGAAGGTATCGAAGTCACGATCGTTCAGAGCAAATGGTCCAAGATTCAGCTGAACGGTAAAGAACACGCACACAGCTTCCGCTATGACGGTCCGGAAACTCGTCGTACTTTCCTGAACTACGACAAGCTGACTAAGAAACTGCAGCTGACCAGCTCTATCAAAGACCTGACCGTGCTGAAATCTACCGGTTCCATGTTTTATGGCTACAACGTGTGTGACTACACCACCCTGCAGCCGACGAAAGATCGCATTCTGTCCACCGACGTTGACGCTTCTTGGACCTTTGATCCGACTCAGATTTCCACCCTGGACGATATCCTGAGCCAACCGAAGCTGTTTGACACCACTTACGATGCCGCGCGTGATGTTACCCTGGAACTGTTTTGTCTGGAAAACAGCCCGTCTGTGCAGGCGACTATGTATAACATGGCTCACAAAATCCTGGAGCAGGTAAAACAGGTGGGCATCGTGACCTACATCCTGCCGAACAAACACTACATCCTGTTCAACCTGGAATGGAAAGGTATTAAAGACAACAAAGAGCTGTTTTACCCGTCTAGCGACCCAAACGGCCTGATTAAATGCACCGTTGGCCGTAAGGGCGACAAAGCGAAATTC
SEQ ID NO:6：
MQSELYSSTYGKANVKFLKVKKDQSNPTVQEILEANVQVLLRGKFEESYTKADNSSIVPTDTVKNTILVEAKNTDVWPIERFAAHLAKHFTSKYGHVEGIEVTIVQSKWSKIQLNGKEHAHSFRYDGPETRRTFLNYDKLTKKLQLTSSIKDLTVLKSTGSMFYGYNVCDYTTLQPTKDRILSTDVDASWTFDPTQISTLDDILSQPKLFDTTYDAARDVTLELFCLENSPSVQATMYNMAHKILEQVKQVGIVTYILPNKHYILFNLEWKGIKDNKELFYPSSDPNGLIKCTVGRKGDKAKF
Example 2 expression of uricase protein of interest
Each of the monoclonal antibodies prepared in step example 1 was picked, inoculated in 100. Mu.g/mL of kana-resistant TB medium, shake-cultured at 37℃at 220rpm until OD600 was between 0.6 and 0.8, induced with IPTG (final concentration of 0.1 mM), placed at 37℃and 18℃respectively, shake-cultured overnight without the addition of IPTG as a control, and cultured at 37℃for 3 hours, and each experiment was repeated. SDS-PAGE identification (35.8 Kda of predicted protein size from Expasy website) was performed by ultrasonication of samples, and the results are shown in FIG. 1 (in the figure, M: maker, NT: control, S: supernatant, P: pellet). Table 2 records the experimental results of protein expression.
TABLE 2
| Bacterial origin | Culture conditions | Soluble protein ratio | Molecular weight of protein |
| Arthrobacter globosus | 37℃ | 90% or more of | 33Kda |
| Arthrobacter globosus | 18℃ | 90% or more of | 33Kda |
| Pseudomonas aeruginosa | 37℃ | Inclusion body expression | 75Kda |
| Pseudomonas aeruginosa | 18℃ | Inclusion body expression | 75Kda |
| Candida | 37℃ | Inclusion body expression | 35Kda |
| Candida | 18℃ | Inclusion body expression | 35Kda |
Example 3 purification of uricase
A strain having a high soluble protein content was selected, and 1.5L of the strain was cultured in a flask in the same manner as in example 2. And centrifugally collecting thalli, wherein the wet weight is as follows: 30g. About 2g of the cells were weighed, and 10ml of Lysis Buffer was added thereto to resuspend on ice. Centrifuging at 20000rpm at 4deg.C for 30min after ultrasonic disruption, collecting supernatant, and filtering with 0.22 μm needle filter to obtain supernatant. Subjecting the supernatant to Ni-column affinity chromatography, subjecting to 0-60% buffer B to linear elution, subjecting the eluate to ion exchange, subjecting to HisTrapTMFF, subjecting to linear elution with 0-60% buffer C to obtain 17.5ml of protein, and subjecting to electrophoresis as shown in figure 2 (M: maker, F: affinity flow-through, F3-G11:0-500mM imidazole linear eluate). The expression level of the target protein was calculated and recorded in table 3. The concentrations of the solutions used are as follows:
Buffer A:50mM Tris、50mM NaCl、5％Glycerol，pH8.0
Buffer B:50mM Tris、50mM NaCl、500mM Imidazole、5％Glycerol，pH8.0
Lysis Buffer:50mM Tris、300mM NaCl、5％Glycerol，pH8.0。
TABLE 3 Table 3
| Bacterial origin | Expression level of target protein |
| Arthrobacter globosus | 1.9196mg/mL |
| Pseudomonas aeruginosa | Inclusion body expression was not purified |
| Candida | Inclusion body expression was not purified |
Example 4 determination of recombinant uricase Activity
The uricase activity prepared in the examples was determined using the Soxhaust kit (cat No. BC 4430). Uricase (1 KU) positive enzyme was purchased from Sigma, dissolved in 0.2mL PBS (50% glycerol) pH 7.4 buffer, and then diluted to 0.01-1U/. Mu.L. The uricase activity measuring working solution is prepared according to the following formulation, and the activity measuring working solution is prepared at present.
1) And adding 6ml of the first reagent for dissolution, and preserving at 4 ℃ for one week.
2) And thirdly, adding 12m1 reagent, dissolving, and preserving at 4 ℃ for two weeks.
3) And the fourth reagent is dissolved by adding 12ml of the first reagent, and is preserved for two weeks at 4 ℃.
4) Reagent five, 12ml of reagent one is dissolved and stored for one week at-20 ℃.
5) The working solution is prepared by uniformly mixing the reagent II, the reagent III, the reagent IV, the reagent V and the reagent VI according to the volume ratio of 1:1:1.1:2, and the working solution is prepared for use.
100. Mu.L of the working solution for measuring activity and 1. Mu.L of positive enzyme at each concentration are added to the ELISA plate, and the reaction is carried out at 25 ℃ for 3min, and the absorbance at 505nm is read. And drawing a standard curve (figure 3) by taking the enzyme concentration as the X axis and the corresponding absorbance value as the Y axis, so that R2 is more than or equal to 0.95. The absorbance was measured by the method described above, and the experiment was repeated 3 times to average 1. Mu.L of uricase prepared in the example. If the average absorbance is within the standard curve range, the enzyme activity is calculated by taking into a linear regression equation. If the concentration is not within the standard curve, the sample is concentrated or diluted and then measured.
The uricase prepared in the example was measured for absorbance as described above, and the results are shown in Table 4 below:
TABLE 4
| Bacterial origin | Enzyme concentration | Average OD505 | Enzyme activity (U/mu L) |
| Arthrobacter globosus | Stock solution | 0.479566667 | 0.061798324 |
| Pseudomonas aeruginosa | Inclusion body | / | / |
| Candida | Inclusion body | / | / |
When the uricase is derived from Arthrobacter sphaeroides, uric acid is used as a catalytic substrate, the activity of the obtained enzyme is 0.062U/. Mu.L, and the concentration of the stock solution is 1.92mg/mL, so that the specific activity is (0.062 multiplied by 1000)/1.92=32.19U/mg.
When the uricase is derived from Pseudomonas aeruginosa and candida, the uricase cannot be folded correctly in a large intestine expression system, and inclusion bodies are formed.
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
Claims (10)
1. A polynucleotide encoding uricase, wherein the polynucleotide is codon optimized and the polynucleotide is selected from any one of the following:
(i) A polynucleotide having a sequence as shown in SEQ ID NO. 1;
(ii) A polynucleotide having a homology of greater than 95% with the sequence shown in SEQ ID NO. 1; and
(iii) Having a polynucleotide complementary to the polynucleotide sequence set forth in (i) or (ii).
2. The polynucleotide according to claim 1, wherein the polynucleotide encoding uricase is obtained by codon optimization of a polynucleotide sequence encoding uricase in Arthrobacter sphaeroides. .
3. An expression vector comprising the polynucleotide of claim 1 or 2.
4. The expression vector of claim 3, wherein the expression vector comprises a polynucleotide sequence that expresses a His x 6 tag.
5. The expression vector according to claim 3, characterized in that it is an E.coli expression vector, preferably pS28a (+).
6. A host cell comprising the expression vector of any one of claims 3 to 5; or alternatively
The polynucleotide according to claim 1 or 2 integrated into the genome of the host cell.
7. A method of preparing uricase, comprising the steps of:
transforming a host cell with a vector comprising the polynucleotide of claim 1;
culturing the host cell to express the uricase.
8. The method according to claim 6, wherein the medium used in culturing the host cell comprises a kanamycin resistance gene.
9. The method of claim 6, wherein the host cell is cultured to express the protein of interest by IPTG induction.
10. A kit, comprising: the polynucleotide of claim 1; or alternatively
The expression vector of any one of claims 3 to 5; or alternatively
The host cell of claim 6; or alternatively
Recombinant uricase prepared according to the method of any one of claims 7-9.
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| CN202210980019.1A CN117587041A (en) | 2022-08-16 | 2022-08-16 | Recombinant uricase, and preparation method and application thereof |
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