Method for constructing a mutant uricase prokaryotic expression engineered bacterium

Abstract

The present invention discloses a method for constructing a mutant uricase prokaryotic expression engineered bacterium, belonging to the field of genetic engineering technology. The present invention performs four active site mutations on a uricase encoding gene derived from Aspergillus flavus to obtain the uricase encoding gene UOX2. A recombinant expression vector for the UOX2 encoding gene is constructed and introduced into Escherichia coli BL21. The resulting uricase-producing prokaryotic expression engineered bacterium is suitable for industrial batch production, and the specific activity of the produced uricase protein reaches 18.3 U / mg, laying a good foundation for large-scale production of uricase.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to a method for constructing a mutant uricase prokaryotic expression engineering bacterium. Background Art

[0002] Uric acid (UA), a metabolite of purine, has extremely low solubility in serum. Uricase (EC1.7.3.3, urate oxidase) is an enzyme that catalyzes the oxidation of uric acid to allantoin. However, during evolution, the gene encoding uricase in primates became pseudogenized, resulting in a lack of functional uricase in humans, making it incapable of converting uric acid. Normal uric acid concentrations are harmless to the human body, but the high-energy, high-purine diet of modern society can easily disrupt the body's uric acid balance, leading to excessive serum uric acid concentrations and hyperuricemia. Therefore, constructing genetically engineered bacteria that produce high levels of uricase and exogenously introducing uricase are of great significance for the treatment of hyperuricemia.

[0003] Currently, uricase genes from various sources have been cloned and expressed. For example, a uricase gene specific to soybean nodules has been cloned. This gene contains approximately 9 bases, with the coding region separated by seven introns, encoding 8 amino acids. Furthermore, a uricase gene from a filamentous fungus has been cloned. This gene encodes 8 amino acids and contains two short introns. In recent years, the main microorganisms used in the commercial production of uricase-producing strains include Aspergillus flavus, Candida utilis, and Bacillus subtilis. Escherichia coli is a genetically engineered host bacterium with a clear genetic background and low cost. Utilizing E. coli and actively developing uricase-encoding genes with higher biological activity has laid a good foundation for the large-scale application of genetically engineered bacteria that produce high uricase production. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a method for constructing a mutant uricase prokaryotic expression engineered bacterium. The present invention mutates the uricase encoding gene sequence from Aspergillus flavus, and optimizes the design based on the genetic codon preference of Escherichia coli and the principle of minimizing the local secondary structure free energy of the prokaryotic translation initiation sequence. UOX2 Encoding genes, based on this, construct UOX2 The recombinant expression vector encoding the gene is introduced into the host cell to construct a prokaryotic expression engineering bacterium that produces uricase.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a uricase mutant, wherein the uricase mutant is obtained by mutating the amino acids 15, 60, 117, and 228 of a wild-type uricase derived from Aspergillus flavus, wherein the amino acid sequence of the wild-type uricase is shown in SEQ ID NO. 1 and is as follows:

[0007] SEQ ID NO.1:

[0008] MSAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLT VLKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASVQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL.

[0009] The nucleotide sequence of the wild-type uricase encoding gene is shown in SEQ ID NO.2, and is as follows:

[0010] SEQ ID NO.2:

[0011] Atgtccgcagtaaaagcagcccgctacggcaaggacaatgtccgcgtctacaaggttcacaaggacgagaagaccggtgtccagacggtgtacgagatgaccgtctgtgtgcttctggagggtgagattgagacctcttacaccaaggccgacaacagcgtcattgtcgcaaccgactccattaagaacaccatttacatcaccgccaagcagaaccccgttactcctcccgagctgttcggctccatcctgggcacacacttcattgagaagtacaaccacatccatgccgctcacgtcaacattgtctgccaccgctggacccggatggacattgacggcaagccacaccctcactccttcatccgcgacagcgaggagaagcggaatgtgcaggtggacgtggtcgagggcaagggcatcgatatcaagtcgtctctgtccggcctgaccgtgctgaagagcaccaactcgcagttctggggcttcctgcgtgacgagtacaccacacttaaggagacctgggaccgtatcctgagcaccgacgtcgatgccacttggcagtggaagaatttcagtggactccaggaggtccgctcgcacgtgcctaagttcgatgctacctgggccactgctcgcgaggtcactctgaagacttttgctgaagataacagtgccagcgtgcaggccactatgtacaagatggcagagcaaatcctggcgcgccagcagctgatcgagactgtcgagtactcgttgcctaacaagcactatttcgaaatcgacctgagctggcacaagggcctccaaaacaccggcaagaacgccgaggtcttcgctcctcagtcggaccccaacggtctgatcaagtgtaccgtcggccggtcctctctgaagtctaaattgtaa。

[0012] Specifically, the uricase mutant is obtained by mutating the 15th amino acid of the wild-type uricase from Aspergillus flavus from arginine to lysine, the 60th amino acid from serine to threonine, the 117th amino acid from histidine to serine, and the 228th amino acid from valine to isoleucine; the amino acid sequence of the uricase mutant is shown in SEQ ID NO. 3, which is as follows:

[0013] MSAVKAARYGKDNVKVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDTIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPSPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLT VLKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASIQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL.

[0014] In a second aspect of the present invention, a gene encoding the above-mentioned uricase mutant is provided, wherein the nucleotide sequence of the encoding gene has four active site mutations, including: a mutation of the 15th amino acid coding sequence CGC to AAG, a mutation of the 60th amino acid coding sequence TCC to ACA, a mutation of the 117th amino acid coding sequence CAC to TCG, and a mutation of the 228th amino acid coding sequence GTG to ATC; the mutated uricase encoding gene is named UOX2 , UOX2 The nucleotide sequence of the coding gene is shown in SEQ ID NO.4, and is as follows:

[0015] atgtccgcagtaaaagcagcccgctacggcaaggacaatgtcAAGgtctacaaggttcacaaggacgagaagaccggtgtccagacggtgtacgagatgaccgtctgtgtgcttctggagggtgagattgagacctcttacaccaaggccgacaacagcgtcattgtcgcaaccgacacaattaagaacaccatttacatcaccgccaagcagaaccccgttactcctcccgagctgttcggctccatcctgggcacacacttcattgagaagtacaaccacatccatgccgctcacgtcaacattgtctgccaccgctggacccggatggacattgacggcaagccaTCGcctcactccttcatccgcgacagcgaggagaagcggaatgtgcaggtggacgtggtcgagggcaagggcatcgatatcaagtcgtctctgtccggcctgaccgtgctgaagagcaccaactcgcagttctggggcttcctgcgtgacgagtacaccacacttaaggagacctgggaccgtatcctgagcaccgacgtcgatgccacttggcagtggaagaatttcagtggactccaggaggtccgctcgcacgtgcctaagttcgatgctacctgggccactgctcgcgaggtcactctgaagacttttgctgaagataacagtgccagcAtCcaggccactatgtacaagatggcagagcaaatcctggcgcgccagcagctgatcgagactgtcgagtactcgttgcctaacaagcactatttcgaaatcgacctgagctggcacaagggcctccaaaacaccggcaagaacgccgaggtcttcgctcctcagtcggaccccaacggtctgatcaagtgtaccgtcggccggtcctctctgaagtctaaattgtaa。

[0016] The active site includes a binding site and a catalytic site.

[0017] In a third aspect, the present invention provides the use of the above encoding gene in constructing a genetically engineered bacterium that produces uricase.

[0018] In a fourth aspect, the present invention provides a prokaryotic expression engineered bacterium that produces uricase, wherein the prokaryotic expression engineered bacterium contains the above-mentioned encoding gene.

[0019] In a fifth aspect, the present invention provides a method for constructing the above-mentioned prokaryotic expression engineering bacteria, comprising the following steps:

[0020] Build UOX2 The recombinant expression vector encoding the gene is introduced into the host cell to construct a prokaryotic expression engineering bacterium that produces uricase.

[0021] The method for constructing the recombinant expression vector is as follows: UOX2 The coding gene was connected to the pET28 (a) plasmid vector to obtain a recombinant expression vector.

[0022] The host cell is Escherichia coli BL21.

[0023] In a fifth aspect, the present invention provides the use of the above-mentioned prokaryotic expression engineered bacteria in the preparation of an enzyme preparation for preventing or treating hyperuricemia.

[0024] The enzyme preparation for preventing or treating hyperuricemia uses the above-mentioned prokaryotic expression engineered bacteria as an active ingredient.

[0025] Preferably, the enzyme preparation is in the form of a powder.

[0026] Beneficial effects of the present invention:

[0027] 1. The present invention subjected the wild-type uricase encoding gene from Aspergillus flavus to four active site mutations, including: mutation of the 15th amino acid encoding sequence CGC to AAG, mutation of the 60th amino acid encoding sequence TCC to ACA, mutation of the 117th amino acid encoding sequence CAC to TCG, and mutation of the 228th amino acid encoding sequence GTG to ATC. The mutated uricase encoding gene was named UOX2 , UOX2 The nucleotide sequence of the coding gene is shown in SEQ ID NO.4, UOX2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3.

[0028] 2. The present invention is constructed by UOX2A recombinant expression vector encoding the gene was constructed and introduced into Escherichia coli BL21. The resulting prokaryotic expression engineered bacteria producing uricase are suitable for industrial mass production, and the specific activity of the produced uricase protein reaches 18.3 U / mg. The preparation method provided by the present invention lays a good foundation for the large-scale application of gene-expressing engineered bacteria with high uricase production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A map was constructed for the recombinant expression vector plasmid pET28(a)-UOX2.

[0030] Figure 2 This is the SDS-PAGE protein electrophoresis result of the sample during the uricase purification process of the present invention. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0032] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present invention, rather than to limit the scope of the present invention.

[0033] Example 1: Mutation of uricase and construction of prokaryotic expression engineered bacteria producing uricase

[0034] 1. Uricase mutation

[0035] The wild-type uricase gene (SEQ ID NO. 2) from A. flavus (Genbank accession number X61766.1) was subjected to four mutations, including: the 15th amino acid coding sequence CGC was mutated to AAG, the 60th amino acid coding sequence TCC was mutated to ACA, the 117th amino acid coding sequence CAC was mutated to TCG, and the 228th amino acid coding sequence GTG was mutated to ATC. The mutated uricase gene was named UOX2 , UOX2 The nucleotide sequence of the coding gene is shown in SEQ ID NO.4.

[0036] 2. Construction of prokaryotic expression engineered bacteria producing uricase

[0037] Synthesize using primers shown in SEQ ID NO.5-SEQ ID NO.6 UOX2 Encoding gene sequences UOX2The coding gene was connected to the pET28 (a) plasmid vector, located between the restriction endonucleases BamHI and XhoI, to obtain the recombinant expression vector plasmid pET28 (a)- UOX2 . The recombinant expression vector plasmid pET28 (a)- UOX2 Transformed into E. coli Top10, containing the recombinant expression plasmid pET28 (a)- UOX2 The top 10 strains were amplified and cultured, and the plasmids were extracted and transformed into Escherichia coli BL21 competent cells to obtain prokaryotic expression engineering bacteria. After picking a single colony and sequencing verification, the prokaryotic expression engineering bacteria that produced uricase were successfully constructed and named Escherichia coli BL21-pET28(a)- UOX2 .

[0038] SEQ ID NO.5( UOX2 -F):GGATCCatgtccgcagtaaaagc;

[0039] SEQ ID NO.6( UOX2 -R):CTCGAGcaatttagacttcagagag.

[0040] Example 2: Prokaryotic expression engineering bacteria BL21-pET28 (a)- UOX2 Inducible expression and protein electrophoresis

[0041] The prokaryotic expression engineering bacteria BL21-pET28 (a) obtained in Example 1 UOX2 To produce the strain, the seeds grown overnight at 37°C and 200 rpm were transferred to LB medium containing kanamycin at a 2% inoculum size and cultured at 37°C and 200 rpm. 600 When the mRNA expression level was 0.8-1.0, the optimal post-induction condition was used, and IPTG with a final concentration of 1 mM was added to the culture medium at 20°C and 200 rpm to induce expression for 6 h.

[0042] After induction, the bacterial pellet was collected by centrifugation at 8000 rpm, and the pellet was resuspended and washed three times with 20 mM Tris-HCl buffer (pH=8.0) at a ratio of 10 times the wet weight of the bacterial cell. After washing, it was resuspended according to the above ratio and disrupted by ultrasound under ice bath conditions (disruption conditions: 450W, supersonication for 3 s, pause for 7 s, for a total of 20 min). The cell disruption solution was centrifuged at 11000 rpm for 10 min and filtered through a 0.22 μm filter membrane to obtain the crude uricase enzyme solution.

[0043] The crude uricase enzyme solution was subjected to protein electrophoresis (SDS-PAGE) to obtain a protein band with a molecular weight of about 38 kDa between 41 kDa and 31 kDa, which was not present in the control group (blank E. coli BL21). The results of SDS-PAGE electrophoresis showed that the prokaryotic expression engineering bacteria BL21-pET28(a)- UOX2 Uricase is expressed.

[0044] Example 3: Purification of crude uricase enzyme solution and activity detection

[0045] To facilitate large-scale purification, the uricase obtained in Example 2 was tagged with a 6-His tag, and the crude uricase enzyme solution was purified using nickel ion affinity chromatography. The specific steps are as follows:

[0046] Take 5 mL Ni-NAT resin in a centrifuge tube, centrifuge at 3000 rpm for 2 min to remove the supernatant;

[0047] The crude uricase solution obtained in Example 2 was mixed with 20 mM imidazole solution at a ratio of 1:1, and twice the volume of the resin was added to the centrifuge tube and mixed thoroughly (rotation and shaking for 60 min), centrifuged, and the supernatant was saved for subsequent analysis; twice the volume of the resin was mixed with 20 mM imidazole solution and the resin was mixed thoroughly, centrifuged, and the supernatant was saved for subsequent analysis, and the washing was repeated, and the absorbance at 280 nm was detected until the concentration of the eluate remained constant; 1 times the volume of the resin was mixed with 500 mM imidazole solution and the resin was mixed thoroughly to elute the His tag, and the supernatant was carefully aspirated and saved. This was repeated three times. The supernatant was the purified product. The SDS-PAGE detection results of the purified product were as shown below. Figure 2 shown.

[0048] Comparative Example 1:

[0049] In this comparative example 1, the wild-type uricase gene sequence shown in SEQ ID NO. 2 was subjected to two mutations, including mutation of the 15th amino acid coding sequence CGC to AAG and mutation of the 60th amino acid coding sequence TCC to ACA. The mutated uricase coding gene was used to construct a prokaryotic expression engineered bacterium according to the method described in Example 1, induced expression was performed according to the description in Example 2, and the crude uricase enzyme solution produced by the engineered bacterium was purified according to the method described in Example 3 to obtain a purified product.

[0050] Comparative Example 2:

[0051] In this comparative example 2, the wild-type uricase gene sequence shown in SEQ ID NO. 2 was subjected to two mutations, including mutation of the amino acid coding sequence CAC at position 117 to TCG and mutation of the amino acid coding sequence GTG at position 228 to ATC. The mutated uricase coding gene was used to construct a prokaryotic expression engineered bacterium according to the method described in Example 1, induced expression was performed according to the description in Example 2, and the crude enzyme solution produced by the engineered bacterium was purified according to the method described in Example 3 to obtain a purified product.

[0052] Comparative Example 3:

[0053] In this comparative example 3, the unmutated wild-type uricase gene sequence shown in SEQ ID NO. 2 was used to construct a prokaryotic expression engineered bacterium according to the method described in Example 1. Expression was induced according to the description in Example 2, and the crude enzyme solution produced by the engineered bacterium was purified according to the method described in Example 3 to obtain a purified product.

[0054] Test Example 1: Uricase Activity Detection in Each Group

[0055] The purified products obtained in Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were collected, and the uricase activity of each group was assayed. The assay principle is as follows: uricase can catalyze the oxidation of uric acid to produce allantoin. The wavelengths of their maximum absorbance differ: uric acid at 293 nm and allantoin at 224 nm. Within a certain range, their concentrations are proportional to their absorbance. The uricase activity was calculated by measuring the absorbance of the reaction system.

[0056] The specific detection process is as follows: preheating uric acid solution (0.001% Triton-X100, 1mM EDTA, 50mM boric acid, 0.001% uric acid, pH = 8.5) to 37°C, mixing 2.5mL of uric acid solution and 0.5mL of uricase solution from Example 3 as test group 1, mixing 2.5mL of uric acid solution and 0.5mL of uricase solution from Comparative Example 1 as test group 2, mixing 2.5mL of uric acid solution and 0.5mL of uricase solution from Comparative Example 2 as test group 3, and mixing 2.5mL of uric acid solution and 0.5mL of uricase solution from Comparative Example 3 as test group 4, and measuring using an ultraviolet spectrophotometer (Shanghai Meixi). The specific activity of the purified proteins in each group was calculated according to enzyme activity (U / mL) = (ΔA293nm×Vt) / (12.04×1.0×Vs×t). The test results are shown in Table 1.

[0057] Where Vt = total volume 3 mL, Vs = sample volume 0.5 mL, t = reaction time 5 min, 12.04 = molar extinction coefficient of uric acid, and 1.0 refers to the cuvette optical diameter (cm).

[0058] Table 1: Uricase activity detection in each group

[0059]

[0060] The test results showed that the present invention produced a synergistic effect after the uricase encoding gene from Aspergillus flavus was mutated at four positions. The prokaryotic expression engineering bacteria BL21-pET28(a)- UOX2 The uricase-producing activity is excellent, and the present invention lays a good foundation for the large-scale application of gene-expressing engineered bacteria with high uricase production.

[0061] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principles of the present application shall be

[0062] Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A uricase mutant, characterized in that: The amino acid sequence of the uricase mutant is shown in SEQ ID NO.

3.

2. A gene encoding the uricase mutant according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

3. Use of the coding gene according to claim 2 in constructing a genetically engineered bacterium that produces uricase.

4. A prokaryotic expression engineered bacterium producing uricase, characterized in that: The prokaryotic expression engineered bacteria contains the coding gene according to claim 2.

5. A method for constructing the prokaryotic expression engineering bacteria according to claim 4, characterized in that: The steps include: Construct a recombinant expression vector encoding the gene according to claim 2, introduce the recombinant expression vector into a host cell, and construct a prokaryotic expression engineered bacterium that produces uricase.

6. The construction method according to claim 5, characterized in that: The method for constructing the recombinant expression vector is: connecting the coding gene described in claim 2 to the pET28 (a) plasmid vector to obtain the recombinant expression vector.

7. The construction method according to claim 5, characterized in that: The host cell is Escherichia coli BL21.

8. Use of the prokaryotic expression engineered bacteria according to claim 4 in the preparation of an enzyme preparation for preventing or treating hyperuricemia.

9. The use according to claim 8, characterized in that The enzyme preparation for preventing or treating hyperuricemia uses the prokaryotic expression engineered bacteria according to claim 4 as an active ingredient.

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