Construction method of mutated uricase prokaryotic expression engineering bacteria

Abstract

The invention discloses a construction method of a mutated uricase prokaryotic expression engineering bacterium, and belongs to the technical field of gene engineering. The uricase encoding gene UOX2 is obtained by mutating four active sites of the uricase encoding gene derived from aspergillus flavus, a recombinant expression vector of the UOX2 encoding gene is constructed and is introduced into escherichia coli BL21, and the constructed prokaryotic expression engineering bacterium for producing uricase is suitable for industrial batch production and has a broad application prospect. And the specific activity of the uricase protein reaches 18.3 U / mg, so that a good foundation is laid for large-scale production of uricase.

Description

Technical Field

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

[0002] Uric acid (UA) is a metabolite of purine, and its solubility in serum is extremely low. Uricase (EC1.7.3.3, urate oxidase) is an enzyme that can catalyze the oxidation of uric acid into allantoin. However, during the evolution process, the gene encoding uricase in primates has undergone pseudogenesis, resulting in the lack of functional uricase in humans and the inability to convert uric acid. Normal uric acid concentration is harmless to the human body, but the high-energy, high-purine diet in modern society can easily disrupt the uric acid balance in the body, leading to excessive uric acid concentration in serum and hyperuricemia. Therefore, constructing genetically expressed engineered bacteria that produce high uricase production and exogenously introducing uricase are of great significance for the treatment of hyperuricemia.

[0003] At present, uricase genes from various sources have been cloned and expressed. For example, the uricase gene unique to soybean nodules has been cloned. This gene contains about 9 bases, and the coding region is separated by seven introns, encoding 8 amino acids. In addition, a uricase gene from filamentous fungi has also been cloned. This gene encodes 8 amino acids and contains two short introns. In recent years, the main microorganisms in the commercial production of uricase-producing strains are Aspergillus flavus, Candida utilis, and Bacillus subtilis. Escherichia coli is a genetic engineering host with a clear genetic background and low cost. The use of Escherichia coli and the active development of uricase encoding genes with higher biological activity have laid a good foundation for the large-scale application of genetically expressed engineering bacteria with 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 engineering bacterium. The present invention mutates the uricase encoding gene sequence derived from Aspergillus flavus, and optimizes the design to obtain the UOX2 encoding gene according to the genetic codon preference of Escherichia coli and the principle of minimizing the local secondary structure free energy of the prokaryotic translation initiation sequence. On this basis, a recombinant expression vector of the UOX2 encoding gene is constructed, and the recombinant expression vector is introduced into a 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 solution:

[0006] In a first aspect, the present invention provides a uricase mutant, wherein the uricase mutant is obtained by mutating the amino acids at positions 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 specifically as follows:

[0007] SEQ ID NO.1:

[0008] MSAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLTV LKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASVQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL*.

[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] MSAVKAARYGKDNVKVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDTIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPSPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLTV LKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASIQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL*.

[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: the 15th amino acid encoding sequence CGC mutates to AAG, the 60th amino acid encoding sequence TCC mutates to ACA, the 117th amino acid encoding sequence CAC mutates to TCG, and the 228th amino acid encoding sequence GTG mutates to ATC; the mutated uricase encoding gene is named UOX2, and the nucleotide sequence of the UOX2 encoding gene is shown in SEQ ID NO.4, which is specifically as follows:

[0015] atgtccgcagtaaaagcagcccgctacggcaaggacaatgtcAAGgtctacaaggttcacaaggacgagaagaccggtgtccagacggtgtacgagatgaccgtctgtgtgcttctggagggtgagattgagacctcttacaccaaggccgacaacagcgtcattgtcgcaaccgacacaattaagaacaccatttacatcaccgccaagcagaaccccgttactcctcccgagctgttcggctccatcctgggcacacacttcattgagaagtacaaccacatccatgccgctcacgtcaacattgtctgccaccgctggacccggatggacattgacggcaagccaTCGcctcactccttcatccgcgacagcgaggagaagcggaatgtgcaggtggacgtggtcgagggcaagggcatcgatatcaagtcgtctctgtccggcctgaccgtgctgaagagcaccaactcgcagttctggggcttcctgcgtgacgagtacaccacacttaaggagacctgggaccgtatcctgagcaccgacgtcgatgccacttggcagtggaagaatttcagtggactccaggaggtccgctcgcacgtgcctaagttcgatgctacctgggccactgctcgcgaggtcactctgaagacttttgctgaagataacagtgccagcAtCcaggccactatgtacaagatggcagagcaaatcctggcgcgccagcagctgatcgagactgtcgagtactcgttgcctaacaagcactatttcgaaatcgacctgagctggcacaagggcctccaaaacaccggcaagaacgccgaggtcttcgctcctcagtcggaccccaacggtctgatcaagtgtaccgtcggccggtcctctctgaagtctaaattgtaa。

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

[0017] The third aspect of the present invention provides the use of the above encoding gene in constructing a genetically engineered bacterium producing 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] A recombinant expression vector of the UOX2 encoding gene is constructed, and the recombinant expression vector is introduced into a host cell to construct a prokaryotic expression engineering bacterium that produces uricase.

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

[0022] The host cell is Escherichia coli BL21.

[0023] The fifth aspect of 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 performs four active site mutations on a wild-type uricase encoding gene derived from Aspergillus flavus, including: the 15th amino acid encoding sequence CGC is mutated to AAG, the 60th amino acid encoding sequence TCC is mutated to ACA, the 117th amino acid encoding sequence CAC is mutated to TCG, and the 228th amino acid encoding sequence GTG is mutated to ATC. The mutated uricase encoding gene is named UOX2, the nucleotide sequence of the UOX2 encoding gene is shown in SEQ ID NO.4, and the amino acid sequence of the protein encoded by the UOX2 gene is shown in SEQ ID NO.3.

[0028] 2. The present invention constructs a recombinant expression vector of the UOX2 encoding gene and introduces the recombinant expression vector into Escherichia coli BL21 to construct a prokaryotic expression engineered bacterium that produces uricase, which is suitable for industrial mass production and has a specific activity of uricase protein of 18.3 U / mg. The preparation method provided by the present invention lays a good foundation for the large-scale application of gene expression engineered bacteria with high uricase production. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0031] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those 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 application, 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. Mutation of uricase

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

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

[0037] The UOX2 coding gene sequence was synthesized using the primers shown in SEQ ID NO.5-SEQ ID NO.6, and the UOX2 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 was transformed into Escherichia coli Top10, and the Top10 strain containing the recombinant expression plasmid pET28(a)-UOX2 was amplified and cultured to extract the plasmid, and the plasmid was transformed into Escherichia coli BL21 competent cells to obtain prokaryotic expression engineering bacteria. After picking a single colony and sequencing verification, a successfully constructed prokaryotic expression engineering bacteria producing uricase was obtained, which was 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: Induced expression and protein electrophoresis of prokaryotic expression engineering bacteria BL21-pET28(a)-UOX2

[0041] The prokaryotic expression engineered bacteria BL21-pET28(a)-UOX2 obtained in Example 1 was used as the production strain. The seeds cultured overnight at 37°C and 200 rpm were transferred into LB medium containing kanamycin at a 2% inoculum and cultured at 37°C and 200 rpm. When the OD 600 When the p-value is 0.8-1.0, the optimal post-induction condition is used, and IPTG with a final concentration of 1 mM is added to the culture medium, and the expression is induced for 6 h at 20°C and 200 rpm.

[0042] After induction, the bacterial precipitate was collected by centrifugation at 8000 rpm, and the precipitate was resuspended and washed three times in 20 mM Tris-HCl buffer (pH = 8.0) at a ratio of 10 times the wet weight of the bacteria. After washing, it was resuspended according to the above ratio and ultrasonically disrupted under ice bath conditions (crushing conditions: 450 W, supersonic 3 s, stop 7 s, a total of 20 min). The cell disruption solution was centrifuged at 11000 rpm for 10 min and filtered through a 0.22 um 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 Escherichia coli BL21). The SDS-PAGE electrophoresis results showed that the prokaryotic expression engineered bacteria BL21-pET28(a)-UOX2 expressed uricase.

[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 6-His, and the crude uricase enzyme solution was purified using nickel ion affinity chromatography. The specific steps are as follows:

[0046] 5 mL of Ni-NAT resin was placed in a centrifuge tube and centrifuged at 3000 rpm for 2 min to remove the supernatant; a 20 mM imidazole solution twice the volume of the resin was fully mixed with the resin to balance the resin, and the supernatant was removed by centrifugation, and the process was repeated twice; the crude uricase solution obtained in Example 2 was mixed with a 20 mM imidazole solution at a ratio of 1:1, and twice the volume of the resin was added to the centrifuge tube and the mixture was fully mixed (rotating and shaking for 60 min), and the mixture was centrifuged, and the supernatant was saved for subsequent analysis; twice the volume of the resin was fully mixed with the 20 mM imidazole solution, and the mixture was 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 unchanged; 1 times the volume of the resin was fully mixed with the resin, the His tag was eluted, and the supernatant was carefully aspirated and saved after centrifugation, and the process was repeated three times. The supernatant was the purified product, and the SDS-PAGE detection result of the purified product was as follows: Figure 2 shown.

[0047] Comparative Example 1:

[0048] In this comparative example 1, two mutations were made to the wild-type uricase gene sequence shown in SEQ ID NO.2, including: the 15th amino acid coding sequence CGC was mutated to AAG and the 60th amino acid coding sequence TCC was mutated to ACA, and the mutated uricase coding gene was used to construct a prokaryotic expression engineering bacterium according to the method described in Example 1, and the expression was induced according to the description of Example 2, and the crude enzyme solution of uricase produced by the engineering bacteria was purified according to the method described in Example 3 to obtain a purified product.

[0049] Comparative Example 2:

[0050] In this comparative example 2, two mutations were made to the wild-type uricase gene sequence shown in SEQ ID NO.2, including: the 117th amino acid coding sequence CAC was mutated to TCG and the 228th amino acid coding sequence GTG was mutated to ATC, and the mutated uricase coding gene was used to construct a prokaryotic expression engineering bacterium according to the method described in Example 1, and the expression was induced according to the description of Example 2, and the crude enzyme solution of uricase produced by the engineering bacteria was purified according to the method described in Example 3 to obtain a purified product.

[0051] Comparative Example 3:

[0052] 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 engineering bacterium according to the method described in Example 1, and the expression was induced according to the description of Example 2, and the crude enzyme solution of uricase produced by the engineering bacterium was purified according to the method described in Example 3 to obtain a purified product.

[0053] Test Example 1: Detection of uricase activity in each group

[0054] After the purified products obtained in Example 4, Comparative Example 1, Comparative Example 2 and Comparative Example 3 were collected, the activity of the uricase obtained in each group was detected. The detection principle is: uricase can catalyze the oxidation of uric acid to generate allantoin, and their maximum absorbance wavelengths are different, uric acid is 293nm, and allantoin is 224nm. Within a certain range, their concentrations are proportional to the absorbance; the activity of uricase is calculated by measuring the absorbance of the reaction system.

[0055] The detection process is specifically as follows: preheating the 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 of Example 4 as test group 1, mixing 2.5mL of uric acid solution and 0.5mL of uricase solution of Comparative Example 1 as test group 2, mixing 2.5mL of uric acid solution and 0.5mL of uricase solution of Comparative Example 2 as test group 3, mixing 2.5mL of uric acid solution and 0.5mL of uricase solution of Comparative Example 3 as test group 4, and measuring using an ultraviolet spectrophotometer (Shanghai Meixi). The specific activity of the purified protein 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.

[0056] Wherein, 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 light path (cm).

[0057] Table 1: Uricase activity detection in each group

[0058] Grouping Vitality Experimental Group 1 18.3 U / mg Experimental Group 2 14.6 U / mg Experimental Group 3 15.2 U / mg Experimental Group 4 11.8 U / mg

[0059] The test results show that after the present invention carried out four mutations in the uricase encoding gene derived from Aspergillus flavus, a synergistic effect was produced, and the constructed prokaryotic expression engineered bacteria BL21-pET28(a)-UOX2 had excellent uricase production activity. The present invention laid a good foundation for the large-scale application of genetic expression engineered bacteria with high uricase production.

[0060] The above description is only the 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, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A uricase mutant, characterized in that: The uricase mutant is obtained by mutating the amino acids at positions 15, 60, 117 and 228 of the wild-type uricase derived from Aspergillus flavus. The amino acid sequence of the wild-type uricase is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene of the wild-type uricase is shown in SEQ ID NO.

2.

2. The uricase mutant according to claim 1, characterized in that The amino acid sequence of the uricase mutant is shown in SEQ ID NO.

3.

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

4.

4. Use of the coding gene according to claim 3 in constructing a genetically engineered bacterium producing uricase.

5. A prokaryotic expression engineered bacterium producing uricase, characterized in that: The prokaryotic expression engineered bacteria contains the coding gene according to claim 3.

6. A method for constructing the prokaryotic expression engineering bacteria according to claim 5, characterized in that: The steps include: Construct a recombinant expression vector of the encoding gene according to claim 3, introduce the recombinant expression vector into a host cell, and construct a prokaryotic expression engineered bacterium that produces uricase.

7. The construction method according to claim 6, characterized in that: The method for constructing the recombinant expression vector is: connecting the coding gene described in claim 3 to the pET28(a) plasmid vector to obtain the recombinant expression vector.

8. The construction method according to claim 6, characterized in that: The host cell is Escherichia coli BL21.

9. Use of the prokaryotic expression engineered bacteria according to claim 5 in the preparation of an enzyme preparation for preventing or treating hyperuricemia.

10. The use according to claim 9, characterized in that: The enzyme preparation for preventing or treating hyperuricemia uses the prokaryotic expression engineered bacteria described in claim 5 as an active ingredient.

Images