Method for improving catalytic activity of heparin 3-O-sulfotransferase 1

Abstract

The invention discloses a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1, and belongs to the technical field of biological engineering. The invention discloses a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1, which comprises the following steps: firstly, selecting escherichia coli as host bacteria, then carrying out expression optimization on the 3-O-sulfotransferase 1 by utilizing a method of fusing an N-terminal with a dissolution promoting label, and further carrying out rational design mutation modification on the optimized 3-O-sulfotransferase 1 sequence to obtain the heparin 3-O-sulfotransferase 1. The high-activity production strain is obtained. The modification of the enzyme has a crucial effect on the anticoagulant activity of heparin, and the improvement of the enzyme activity has positive significance on the enzymatic synthesis rate and the anticoagulant activity of heparin.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and more particularly to a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1. Background Art

[0002] In nature, heparin is a type of polysaccharide synthesized by the Golgi apparatus of eukaryotic cells and secreted outside the cell. It is named because it was first discovered in the liver. It is also present in the lungs, blood vessel walls, intestinal mucosa and other tissues of animals. It is functionally used to interact with various proteins in the extracellular matrix, and plays an important role in anticoagulation, viral invasion, tumor spread and signal transduction. It has excellent anticoagulant effects both in vivo and in vitro and is widely used in clinical practice.

[0003] In terms of chemical structure, it is a polysaccharide chain composed of multiple sulfonated glucosamine, L-iduronide, N-acetylglucosamine and D-glucuronic acid. It is a straight-chain negatively charged linear polysaccharide formed by a series of deacetylation, isomerization and sulfation reactions of the heparin precursor formed by alternating glucuronic acid and N-acetylglucosamine units.

[0004] The mechanism of action of heparin is that the heparin-specific pentasaccharide structure binds to antithrombin III.

[0005] Heparin can bind to antithrombin-Ⅲ (AT-Ⅲ) to enhance its activity, inhibit the formation of thrombin prokinase, and can interfere with the function of thrombin by combining with thrombin and antithrombin to form a complex, prevent the activation of coagulation factors, and prevent platelet aggregation, etc. It is mainly used clinically for thromboembolic diseases, myocardial infarction, cardiovascular surgery, cardiac catheterization, extracorporeal circulation, hemodialysis, etc., and with the progress of pharmacology and clinical medicine, the application of heparin is still expanding.

[0006] At present, the main methods for obtaining heparin are biological extraction, chemical synthesis, chemical enzyme synthesis, and enzymatic synthesis. The biological extraction method is mainly extracted from the small intestinal mucosa of animals, but the heparin obtained by this method often cannot completely remove other glycosaminoglycan impurities, such as chondroitin sulfate, etc. These impurities can cause human immune response, making the obtained product have certain deficiencies in biosafety. Heparin contamination incidents have occurred that endanger patients using the drug; the steps of chemical synthesis of heparin are very cumbersome, and therefore the price is relatively expensive; the difference between chemical enzyme method and enzymatic synthesis is mainly that the first step of the N-deacetylation process uses alkaline reagents or deacetylases. The heparin product synthesized by biological enzyme method has a more uniform structure, higher biosafety, and is simpler and easier than chemical synthesis. Bioenzymatic synthesis of heparin is a way to obtain heparin with better structural uniformity and higher biosafety.

[0007] There are 7 subtypes of 3-O-sulfotransferase in nature. They can be classified into gD type, which can catalyze the substrate to form the glycoprotein binding receptor site of herpes simplex virus type 1 (HSV-1), and AT type, which can catalyze the substrate to form heparan sulfate with anticoagulant function. AT type includes two subtypes, 3-O-sulfotransferase 1 and 3-sulfotransferase 5. This type of enzyme preferentially modifies the aminoglucose whose non-reducing end is a modified glucuronic acid that has not been subjected to C5 isomerase and 2-O-sulfotransferase. Among them, 3-O-sulfotransferase 5 has the functions of both gD type and AT type 3-O-sulfotransferase, while 3-O-sulfotransferase 1 can only specifically catalyze the substrate polysaccharide to form heparin with anticoagulant activity. The activity of 3-O-sulfotransferase 1 in forming anticoagulant structural units is 250 times higher than that of 3-O-sulfotransferase 5. The human heparin 3-O-sulfotransferase 1 enzyme consists of 307 amino acids, with a spatial structure that is approximately spherical and has an obvious substrate binding pocket for binding to the substrate heparin polysaccharide. Heparin 3-O-sulfotransferase 1 catalyzes the sulfonation of the 3-OH of the N-acetylglucosamine unit to form an N-acetylglucosamine unit with a 3-sulfonic acid group. In the chemical structure of heparin, the sulfonic acid group at this position plays a critical role in the realization of the anticoagulant function of heparin. It is currently reported that the sulfonate transfer activity of heparin 3-O-sulfotransferase 1 expressed in Escherichia coli Rosetta 2 cells is 14 pmol / μg. Given that the degree of sulfonation at the 3-OH site has a nearly decisive influence on the activity of heparin, the enzymatic preparation efficiency of heparin is limited under the catalysis of the wild-type 3-OST-1 enzyme with low catalytic activity, which is not conducive to industrial production.

[0008] Therefore, providing a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1 is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0009] In view of this, the present invention provides a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] A heparin 3-O-sulfotransferase 1 mutant is obtained by fusing a solubility-promoting tag MBP to the N-terminus of the heparin 3-O-sulfotransferase 1 and then performing molecular modification to obtain the heparin 3-O-sulfotransferase 1 mutant; the molecular modification comprises any one of (1) to (3):

[0012] (1) The amino acid 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine;

[0013] (2) mutating amino acid 144 of heparin 3-O-sulfotransferase 1 from histidine to arginine;

[0014] (3) mutating the amino acid at position 72 of heparin 3-O-sulfotransferase 1 from tryptophan to arginine, and mutating the amino acid at position 144 from histidine to arginine;

[0015] The amino acid sequence of the heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.2; the amino acid sequence of the MBP is shown in SEQ ID NO.7.

[0016] Further, a gene encoding the heparin 3-O-sulfotransferase 1 mutant.

[0017] Further, a plasmid containing the gene.

[0018] Furthermore, a genetically engineered bacterium is provided for expressing the heparin 3-O-sulfotransferase 1 mutant.

[0019] Furthermore, a method for producing heparin 3-O-sulfotransferase 1 is provided, which uses the genetically engineered bacteria as a production strain to induce fermentation to produce heparin 3-O-sulfotransferase 1.

[0020] Furthermore, a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1 is provided, firstly, a solubility-promoting tag MBP is fused to the N-terminus of heparin 3-O-sulfotransferase 1, and then a heparin 3-O-sulfotransferase 1 mutant is obtained by molecular modification; the molecular modification comprises any one of (1) to (3):

[0021] (1) The amino acid 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine;

[0022] (2) mutating amino acid 144 of heparin 3-O-sulfotransferase 1 from histidine to arginine;

[0023] (3) mutating the amino acid at position 72 of heparin 3-O-sulfotransferase 1 from tryptophan to arginine, and mutating the amino acid at position 144 from histidine to arginine;

[0024] The amino acid sequence of the heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.2; the amino acid sequence of the MBP is shown in SEQ ID NO.7.

[0025] Furthermore, the heparin 3-O-sulfotransferase 1 mutant is used to improve the activity of heparin 3-O-sulfotransferase 1.

[0026] Furthermore, the heparin 3-O-sulfotransferase 1 mutant is used in the fields of food, chemical industry or pharmaceutical industry.

[0027] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for improving the catalytic activity of heparin 3-O-sulfotransferase 1, which has the following beneficial effects:

[0028] The present invention is the first to co-express the chaperone protein plasmid pGro7 with the pMal-c5E vector carrying the human heparin 3-O-sulfotransferase 1 gene in Escherichia coli OrigamiB (DE3) microbial cells, and the obtained enzyme catalytic activity is 15.60U / L, the specific enzyme activity is 1.68U / mg, and the sulfotransfer rate is 12.98pmol / μg.

[0029] The present invention uses Escherichia coli OrigamiB (DE3) microbial cells for the first time to co-express a chaperone protein with 3-O-sulfotransferase 1 after the amino acid at position 72 is mutated from tryptophan to arginine, and the resulting enzyme catalytic activity is 26.40 U / L, which is 69% higher than that before the mutation.

[0030] The present invention uses Escherichia coli OrigamiB (DE3) microbial cells for the first time to co-express a chaperone protein with 3-O-sulfotransferase 1 after the amino acid at position 144 is mutated from histidine to arginine, and the resulting enzyme catalytic activity is 17.21 U / L, which is 11% higher than that before the mutation.

[0031] The present invention adopts Escherichia coli OrigamiB (DE3) microbial cells for the first time to co-express a chaperone protein with a 3-O-sulfotransferase 1 after the amino acid at position 72 is mutated from tryptophan to arginine and the amino acid at position 144 is mutated from histidine to arginine. The obtained enzyme catalytic activity is 34.41 U / L, which is 121% higher than that before mutation. The specific enzyme activity is 2.40 U / mg, which is 43% higher than that of the wild type. The sulfotransfer rate is 23.61 pmol / μg, which is 82% higher than that of the wild type.

[0032] The present invention integrates the linearized plasmid of the pPICZαA expression vector carrying the 3-O-sulfotransferase 1 gene into the genome of the Pichia pastoris GS115 microbial cell for the first time, and the obtained enzyme catalytic activity is 66.77 U / L.

[0033] The present invention firstly expresses 3-O-sulfotransferase 1 in which amino acid 72 is mutated from tryptophan to arginine in Pichia pastoris GS115 microbial cells, and the obtained enzyme catalytic activity is 70.27 U / L, which is 5% higher than that before the mutation.

[0034] The present invention firstly expresses 3-O-sulfotransferase 1 in which amino acid 144 is mutated from tryptophan to arginine in Pichia pastoris GS115 microbial cells, and the obtained enzyme catalytic activity is 68.86 U / L, which is 3% higher than that before the mutation.

[0035] The present invention is the first to express 3-O-sulfotransferase 1 with N-terminal fused Saccharomyces cerevisiae codon-optimized MBP protein in Pichia pastoris GS115 microbial cells, and the obtained enzyme catalytic activity is 75.41 U / L, which is 13% higher than that before the fusion tag.

[0036] The present invention is the first to express the 3-O-sulfotransferase 1 in which the amino acid at position 72 is mutated from tryptophan to arginine at the N-terminus of the Saccharomyces cerevisiae codon-optimized MBP protein in Pichia pastoris GS115 microbial cells, and the obtained enzyme catalytic activity is 88.51 U / L, which is 17% higher than the wild-type enzyme fused with a fusion-promoting tag and 33% higher than the wild-type enzyme without a fusion tag. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0038] Figure 1 The accompanying drawing is a schematic diagram of the catalysis of heparin 3-O-sulfotransferase 1 of the present invention;

[0039] Figure 2 The accompanying drawing is a plasmid map of the N-terminal fusion solubility-promoting tag MBP of heparin 3-O-sulfotransferase 1 of the present invention;

[0040] Figure 3 The accompanying figure is an SDS-PAGE of heparin 3-O-sulfotransferase 1 expressed in Escherichia coli OrigamiB (DE3) after N-terminal fusion of MBP tag;

[0041] Wherein, M: maker; Lane 1: control strain intracellular supernatant; Lane 2: wild-type heparin 3-O-sulfotransferase 1 intracellular supernatant in Escherichia coli; Lane 3: heparin 3-O-sulfotransferase 1 (W72R) intracellular supernatant in Escherichia coli; Lane 4: heparin 3-O-sulfotransferase 1 (H144R) intracellular supernatant in Escherichia coli; Lane 5: heparin 3-O-sulfotransferase 1 (W72R / H144R) intracellular supernatant in Escherichia coli; Lane 6: wild-type heparin 3-O-sulfotransferase 1 pure enzyme; Lane 7: heparin 3-O-sulfotransferase 1 (W72R / H144R) pure enzyme;

[0042] Figure 4 The accompanying drawings show the interaction between the sites to be mutated in 3-OST-1 and the corresponding sites in 3-OST-3 of the present invention and the substrate;

[0043] Figure 5 The accompanying drawings show the enzymatic activity of heparin 3-O-sulfotransferase 1 and its mutants expressed in Escherichia coli OrigamiB (DE3);

[0044] Figure 6 The attached figure shows the enzymatic activity of heparin 3-O-sulfotransferase 1 and its mutants expressed in Pichia pastoris GS115. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Method for determining the activity of crude 3-OST-1 enzyme solution: Sodium heparin from porcine small intestine was used as a substrate to construct a catalytic reaction system, which included: 50 mM Tris-HCl (pH = 7.4), 50 mM MES, 50 mM PNPS (p-nitrobenzenesulfonic acid), 0.5 mM PAP (3',5'-adenosine diphosphate), 2 mg / mL ASTIV purified enzyme (the gene was synthesized by the company), 1 mM CaCl2, 500 μL / mL of the crude 3-OST-1 (heparin 3-O-sulfotransferase 1) enzyme solution prepared in each embodiment, 2 mg / mL sodium heparin from porcine small intestine (purchased from Sangon Biotechnology Co., Ltd.), react at 40°C for 2 hours, then heat at 90°C for 10 minutes to terminate the reaction, and measure the absorbance at a wavelength of 410 nm.

[0047] The definition of 3-OST-1 enzyme activity is the amount of enzyme required to generate 1 μmol PNP (p-nitrophenol) per minute at 40°C and pH 7.4.

[0048] The schematic diagram of the reaction of 3-OST-1 enzyme catalyzing the modification of substrate polysaccharide into heparin is shown in Figure 1 .

[0049] Example 1 Preparation of purified enzymes of 3-OST-1 and ASTIV (acylsulfate transferase IV) in Escherichia coli

[0050] The nucleotide sequence of the gene encoding Escherichia coli codon-optimized heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.1.

[0051] CGTCCTGCAGAACTGGGCCAGCAGGAACTGCTGCGTAAAGCAGGTACCCTGCAGGATGATGTGCGTGATGGTGTTGCCCCGAATGGCAGTGCACAGCAGCTGCCGCAGACCATTATTATTGGTGTGCGTAAAGGTGGCACCCGCGCACTGCTGGAAATGCTGAGTCTGCATCCGGATGTTGCAGCAGCAGAAAATGAAGTTCATTTCTTTGATTGGGAAGAACATTATAGCCATGGTCTGGGTTGGTATCTGAGCCAGATGCCGTTTAGCTGGCCGCATCAGCTGACCGTGGAAAAAACCCCGGCCTATTTTACCAGTCCGAAAGTGCCGGAACGTGTTTATAGTATGAATCCGAGCATTCGTCTGCTGCTGATTCTGCGCGATCCGAGCGAACGTGTGCTGAGTGATTATACCCAGGTGTTTTATAATCATATGCAGAAACATAAGCCGTATCCGAGCATTGAAGAATTTCTGGTGCGCGATGGTCGTCTGAATGTGGATTATAAAGCACTGAATCGCAGTCTGTATCATGTTCATATGCAGAATTGGCTGCGTTTCTTTCCGCTGCGTCATATTCATATTGTTGATGGCGATCGCCTGATTCGCGATCCGTTTCCGGAAATTCAGAAAGTTGAACGCTTTCTGAAACTGAGTCCGCAGATTAATGCAAGCAATTTTTATTTTAACAAGACCAAGGGCTTCTATTGTCTGCGTGATAGTGGCCGTGATCGTTGTCTGCATGAAAGTAAAGGTCGCGCACATCCGCAGGTGGATCCGAAACTGCTGAATAAGCTGCATGAATATTTTCATGAACCGAATAAGAAGTTCTTCGAACTGGTTGGCCGCACCTTTGATTGGCATTAA; SEQ ID NO.1。

[0052] Heparin 3-O-sulfotransferase 1 is derived from Homo, and its amino acid sequence is shown in SEQ ID NO.2.

[0053] RPAELGQQELLRKAGTLQDDVRDGVAPNGSAQQLPQTIIIGVRKGGTRALLEMLSLHPDVAAAENEVHFFDWEEHYSHGLGWYLSQMPFSWPHQLTVEKTPAYFTSPKVPERVYSMNPSIRLLLILRDPSERVLSDYTQVFYNHM SEQ IDNO.2.

[0054] 1) Construction of recombinant Escherichia coli expressing ASTIV and 3-OST-1

[0055] The ASTIV gene (Gene ID: 83783) synthesized by the company was ligated with the pET-19b vector linearized by endonucleases NdeI and BamHI using T4 ligase (Thermo Scientific) according to the instructions to obtain the recombinant plasmid pET-19b-ASTIV; the plasmid pET-19b-ASTIV was transformed into the E. coli BL21 (DE3) host bacteria to obtain the recombinant E. coli BL21 (DE3)-pET-19b-ASTIV expressing ASTIV.

[0056] The gene of the Escherichia coli codon-optimized 3-OST-1 enzyme synthesized by the company (the gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) was connected to the pMal-c5E linear vector by seamless cloning to obtain the recombinant plasmid pMal-c5E-3-OST-1. The plasmid map is shown in Figure 2 .

[0057] The method for preparing the pMal-c5E linear vector is as follows: using primers to PCR amplify the pMal-c5E vector (carrying MBP) template to obtain the pMal-c5E linear vector; the primer sequences are as follows:

[0058] F1: 5'-GATTGGCATTAAAAGCTTCAAATAAAACGAAAGGCTCAG-3'; SEQ ID NO.3;

[0059] R1: 5'-TCTGCAGGACGATGATGATGATGATGATGCATATGCG-3'; SEQ ID NO.4.

[0060] PCR reaction program: 98℃3min; 98℃10s, 56℃15s, 72℃80s, 34cycles; 72℃10min; 12℃hold), PCR used Primestar enzyme (Takara), PCR system was in accordance with the reagent instructions. The connection method was to mix the linear vector and 3-OST-1 gene and connect them using a seamless cloning kit (Beijing Quanshijin Biotechnology Co., Ltd.), and the conditions and proportions were in accordance with the instructions. The recombinant plasmid pMal-c5E-3-OST-1 500ng and the chaperone protein plasmid pGro7 (purchased from Beijing Huayueyang Biological Co., Ltd.) 500ng were added to E. coli OrigamiB (DE3) competent cells (Beijing Huayueyang Biological Co., Ltd.), and the two plasmids were transformed into E. coli OrigamiB (DE3) according to the method described in the chemical transformation manual to obtain recombinant E. coli OrigamiB (DE3)-pMal-c5E-3-OST-1 expressing 3-OST-1. The pGro7 and pMal-c5E empty plasmids were co-transformed into E. coli OrigamiB (DE3) competent cells according to the same operation as above to obtain the control group strain.

[0061] 2) Inducible expression of recombinant Escherichia coli expressing ASTIV and 3-OST-1

[0062] Pick a single colony of recombinant E. coli BL21 (DE3) - pET-19b - ASTIV in 4 mL LB medium containing 100 μg / mL ampicillin, and culture it overnight at 37 ° C, 220 rpm. Transfer 1 mL of bacterial solution from the test tube to 50 mL LB medium with the same antibiotic concentration as the test tube, and culture it at 37 ° C, 200 rpm for about 3 h until OD 600 nm was between 0.8-1.0, IPTG was added at a final concentration of 0.5 mM, and induced at 16°C for 22 h.

[0063] Single colonies of recombinant E. coli OrigamiB(DE3)-pMal-c5E-3-OST-1 and control strains were picked and cultured in 4 mL LB medium containing 100 μg / mL ampicillin, 30 μg / mL chloramphenicol and 50 μg / mL kanamycin at 37°C, 200 rpm overnight. 1 mL of bacterial solution was transferred from the test tube to 50 mL LB medium with the same antibiotic concentration as the test tube, and cultured at 37°C, 200 rpm for about 3 h until OD 600 nm was between 0.8-1.0, IPTG was added at a final concentration of 0.5 mM, and induced at 16°C for 22 h.

[0064] The above cultures were centrifuged at 4000 rpm and 4°C for 5 min to collect the cells, and the cells were resuspended in Tris-HCl Buffer with 1 / 4 of the original bacterial solution volume. The cells were broken by a high-pressure homogenizer at 800-1000 Bar, and centrifuged at 4000 rpm for 10 min to remove cell debris. The supernatant crude enzyme solution was collected. The supernatant crude enzyme solution of the control strain was analyzed by SDS-PAGE. Figure 3 Lane 1, SDS-PAGE of wild-type 3-OST-1 supernatant crude enzyme solution Figure 3 Middle lane 2. The crude enzyme solution containing 3-OST-1 was used to directly test the 3-OST-1 enzyme activity, or to purify 3-OST-1 to obtain pure enzyme for SDS-PAGE test; the crude enzyme solution containing ASTIV was purified to obtain ASTIV purified enzyme and added to the 3-OST-1 activity test reaction system to complete the activity test. The enzyme catalytic activity of wild-type 3-OST-1 expressed by E. coli OrigamiB (DE3) was 15.605U / L.

[0065] 3) Purification of ASTIV and 3-OST-1 enzymes

[0066] Tris-HCl buffer (50mM Tris, 300mM NaCl, 20% glycerol, pH = 7.4) was passed through a pre-cooled Ni affinity chromatography column. The crude enzyme solution was repeatedly passed through the agarose filler and then the column was slowly washed with 5 column volumes of Tris-HCl buffer. Contaminants were washed away with 6-7 column volumes of Wash Buffer (50mM Tris, 300mM NaCl, 30mM imidazole, pH = 7.4). The target protein was eluted with Elution buffer (50mM Tris, 300mM NaCl, 250mM imidazole, 20% glycerol, pH = 7.4) and collected. 1L E.coli BL21(DE3)-pET-19b-ASTIV bacterial solution can be purified and concentrated to obtain 10mL of pure enzyme with a concentration of 8-12mg / mL, and 1L E.coli OrigamiB(DE3)-pMal-c5E-3-OST-1 bacterial solution can be purified and concentrated to obtain 1mL of 0.35mg / mL pure enzyme. SDS-PAGE of 3-OST-1 wild-type pure enzyme is shown in Figure 3 Middle lane 6.

[0067] In the method for determining the activity of 3-OST-1 crude enzyme solution, 500 μL / mL crude enzyme solution was replaced with 8 μg / mL 3-OST-1 pure enzyme for reaction. The method for determining the sulfo group transfer rate was to calculate the amount of PNP generated by each μg enzyme transfer sulfo group, and the method for determining the specific enzyme activity was to calculate the sulfo group transfer activity per mg of 3-OST-1 enzyme. The sulfo group transfer rate of 3-OST-1 wild-type pure enzyme was 12.98 pmol / μg, and the specific enzyme activity was 1.68 U / mg.

[0068] Example 2 Preparation of Heparin 3-OST-1 Enzyme in Pichia pastoris

[0069] The nucleotide sequence of the gene encoding the Pichia pastoris codon-optimized heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.5.

[0070] AGACCAGCTGAATTGGGTCAACAAGAATTGTTGAGAAAGGCCGGTACTTTGCAAGATGATGTTAGAGATGGTGTTGCTCCAAACGGTTCTGCTCAACAATTGCCACAAACTATTATTATCGGTGTCCGTAAGGGTGGTACTCGTGCTTTGTTGGAAATGTTGTCTTTGCATCCAGATGTCGCCGCTGCTGAAAACGAAGTTCATTTTTTTGATTGGGAGGAGCACTACTCCCACGGTTTGGGTTGGTACTTGTCTCAAATGCCATTTTCTTGGCCACATCAATTGACTGTTGAAAAGACTCCAGCTTACTTTACCTCTCCAAAGGTTCCAGAAAGAGTTTACTCTATGAACCCATCTATTCGCTTGTTGTTGATCTTGCGTGACCCATCTGAGAGAGTTTTGTCTGACTACACTCAAGTTTTTTACAACCACATGCAGAAGCACAAGCCATACCCATCTATTGAAGAATTTTTGGTCCGTGACGGTCGTTTGAACGTTGATTACAAGGCTTTGAACAGATCCTTGTACCATGTTCATATGCAGAACTGGTTGCGTTTTTTTCCATTGAGACATATCCACATCGTCGATGGTGACAGATTGATTCGTGATCCATTTCCAGAGATCCAAAAGGTTGAAAGATTTTTGAAGTTGTCCCCACAGATCAACGCTTCCAACTTTTACTTTAACAAGACCAAGGGCTTCTACTGCTTGCGTGATTCTGGTAGAGATAGATGTTTGCATGAATCCAAGGGTAGAGCTCATCCACAAGTTGATCCAAAGTTGTTGAACAAGTTGCACGAATACTTTCACGAGCCAAACAAGAAGTTCTTTGAATTGGTTGGTCGTACCTTCGACTGGCAT; SEQ ID NO.5。

[0071] The nucleotide sequence encoding the MBP gene optimized for Saccharomyces cerevisiae codons is shown in SEQ ID NO.6.

[0072]

[0073] MBP is derived from Escherichia coli (strain K12), and the amino acid sequence is shown in SEQ ID NO.7.

[0074] MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQS GLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYE SEQ ID NO.7.

[0075] 1) Construction of recombinant Pichia pastoris expressing 3-OST-1

[0076] The Pichia pastoris codon-optimized 3-OST-1 gene (SEQ ID NO.5) synthesized by the company was connected to the pPICZαA vector by seamless cloning, wherein the pPICZαA vector was pre-digested with EcoRI and XbaI restriction endonucleases, and the connection method was the same as in Example 1 to obtain the recombinant plasmid pPICZαA-3-OST-1; the AOX1 region of the recombinant plasmid was linearized by single digestion with PmeI and then electroporated into Pichia pastoris GS115, and the successful transfer of the target fragment was verified by PCR. By setting different BleoR resistance concentrations of 5 mg / mL, 8 mg / mL, 12 mg / mL and 20 mg / mL, a high copy number of the target strain was screened and integrated to obtain the recombinant strain P. pastoris GS115-pPICZαA-3-OST-1 that can grow in a high resistance concentration environment was obtained.

[0077] The gene of the codon-optimized MBP (maltose binding protein) of Saccharomyces cerevisiae synthesized by the company (the gene sequence is shown in SEQ ID NO.6, and the amino acid sequence is shown in SEQ ID NO.7) was connected to the 3-OST-1 gene (SEQ ID NO.5), and connected with the pPICZαA vector, wherein the MBP, 3-OST-1, and pPICZαA fragments are assembled by a seamless cloning method, wherein the pPICZαA vector is pre-digested with EcoRI and XbaI restriction endonucleases to obtain a recombinant plasmid pPICZαA-MBP-3-OST-1; the AOX1 region of the recombinant plasmid pPICZαA-MBP-3-OST-1 is linearized by single digestion with PmeI, and then electroporated into Pichia pastoris GS115, and the successful transfer of the target fragment is verified by PCR, and a target strain with a high copy number is obtained by screening and integration by setting different BleoR resistance concentrations of 5 mg / mL, 8 mg / mL, 12 mg / mL and 20 mg / mL, and a recombinant strain P. pastoris GS115-pPICZαA-MBP-3-OST-1 that can grow in a high resistance concentration environment is obtained.

[0078] 2) Expression of 3-OST-1 and MBP-3-OST-1 in recombinant Pichia pastoris

[0079] Pick a single colony of recombinant Pichia pastoris GS115-pPICZαA-3-OST-1 or P. pastoris GS115-pPICZαA-MBP-3-OST-1 into 5 mL YPD medium and culture overnight at 30°C and 200 rpm. Transfer the bacterial solution from the test tube to 50 mL BMGY medium to make the initial OD 600 nm was 0.1, and cultured at 30°C, 200 rpm for about 12-14 h until OD 600 nm was about 12. The cells were collected and transferred to BMMY medium and cultured at 28°C for 96 h, with 1% methanol added every 24 h.

[0080] The above culture was centrifuged at 4000 rpm and 4°C for 10 min to collect the bacteria, resuspended in Tris-HCl Buffer, and disrupted by a high-pressure homogenizer at 1600-1800 Bar. The cells were centrifuged at 4000 rpm for 20 min to remove cell debris, and the supernatant crude enzyme solution was collected. The crude enzyme solution containing 3-OST-1 was directly used for 3-OST-1 enzyme activity test.

[0081] Example 33 - Molecular modification of OST-1 enzyme

[0082] 1) 3-OST-1 mutant screening method

[0083] In the molecular three-dimensional structure display software PyMOL, the spatial structural homology of the human 3-OST-1 / PAP binary complex (PDB ID: 1ZRH) and the human 3-OST-3 / PAP / tetrasaccharide heparin substrate ternary complex (PDB ID: 1T8U) enzyme molecular crystal structures were overlapped to find the important residues for the interaction between the two subtypes of 3-OST enzymes and the substrates. Single-point saturation mutations were performed on the sites in BIOVIADiscovery Studio, and the stability of the mutations was determined based on the mutation energy. The mutation strategy was designed based on the spatial structure-activity relationship.

[0084] The specific method is to overlap the spatial structures of the two enzyme-substrate crystal structure models in the molecular three-dimensional structure display software PyMOL, and find residues that have significant differences in substrate interaction, and where 3-OST-1 interacts weakly with the substrate sugar chain at this position while 3-OST-3 enzyme has a stronger interaction at this site. The most appropriate mutation strategy is selected for each residue to be optimized in the 3-OST-1 enzyme structure among 20 common amino acids. During the design process, the strategy should be formulated based on factors such as the spatial size of the position, the spatial orientation of the polar group, the strength of the polarity, and the interaction relationship with other functional groups of the substrate. Take the interaction distance between the two polar atoms in The interaction is stronger within The interaction is weaker than The interactions within the range are considered to be negligible.

[0085] like Figure 4 Trp72 of the 3-OST-1 enzyme (corresponding to amino acid number Trp59 in 1ZRH) is too far away from the 2-O-sulfonic acid group on the 2-idurose ring to interact, while the corresponding 3-OST-3 enzyme Arg190 has a longer spatial shape and stronger polarity, and the interaction distance with this group is Therefore, the position can be changed to Arg like in 3-OST-3 enzyme to increase its spatial length and polarity. The imidazole group of 3-OST-1 enzyme His144 (corresponding to the amino acid number His131 in 1ZRH) is far away from the 1-hydroxyl group of glucosamine unit 1 and has no interaction. The interaction distance between Arg260 of 3-OST-3 enzyme and this group is Therefore, it was considered to mutate this site to Arg, which is more sterically hindered and more polar.

[0086] 2) Preparation method of 3-OST-1 mutant

[0087] The method for 72-site mutation in E. coli is to use primers F2, R2 and F3, R3 to perform PCR amplification on the E. coli codon preference 3-OST-1 gene (SEQ ID NO. 1), respectively, with the same procedure and system as in Example 1, to obtain gene fragments gene1 and gene2.

[0088] F2: 5'-CATCATCATCGTCCTGCAGAACTGGGC-3'; SEQ ID NO.8;

[0089] R2: 5'-CCAGACCATGGCTATAATGTTCTTCACGATCAAAGAAAT-3'; SEQ ID NO.9;

[0090] F3: 5'-GAAGAACATTATAGCCATGGTCTGGGT-3'; SEQ ID NO.10;

[0091] R3: 5'-AAGCTTTTTAATGCCAATCAAAGGTGCG-3'; SEQ ID NO. 11.

[0092] According to the system (primestar 25μL, gene11μL, gene21μL, ddH2O 23μL) and procedure (98℃3min; 98℃10s, 56℃15s, 72℃80s, 5cycles; 72℃10min12℃hold), 2μL of primers F2 and R3 were added to the PCR product, and then the PCR reaction was carried out according to the PCR reaction in Example 1 to obtain gene 3-OST-1 (W72R).

[0093] The method for mutation of site 144 in E. coli is to use primers F2, R4 and F4, R3 to perform PCR amplification on the E. coli codon preference 3-OST-1 gene (SEQ ID NO. 1), respectively, with the same procedure and system as in Example 1, to obtain gene fragments gene3 and gene4.

[0094] F4: 5'-TGCAGAAACATAAGCCGTATCCGAG-3'; SEQ ID NO. 12;

[0095] R4: 5'-CGGATACGGCTTATGTTTCTGCATACGATTATAAAACACC-3'; SEQ ID NO. 13.

[0096] According to the system (primestar 25 μL, gene31 μL, gene41 μL, ddH2O 23 μL) and the above-mentioned procedure, 2 μL of primers F2 and R3 were added to the PCR product, and then the PCR reaction was carried out according to the PCR reaction in Example 1 to obtain gene 3-OST-1 (H144R).

[0097] The 72 and 144 double-site mutation method in Escherichia coli uses the Escherichia coli codon-preferred gene 3-OST-1 (W72R) as a template and performs mutation according to the same steps as the 144-site mutation method to obtain the gene 3-OST-1 (W72R / H144R).

[0098] The plasmid construction, induced expression and activity test methods were the same as those in Example 1. SDS-PAGE of the crude enzyme solution of mutant 3-OST-1 (W72R) Figure 3 Middle lane 3: SDS-PAGE of crude enzyme solution of mutant 3-OST-1 (H144R) Figure 3 Middle lane 4: SDS-PAGE of crude enzyme solution of mutant 3-OST-1 (W72R / H144R) Figure 3 Middle lane 5: pure enzyme SDS-PAGE of mutant 3-OST-1 (W72R / H144R) Figure 3 Middle lane 7.

[0099] The construction of the mutant gene in Pichia pastoris was performed by PCR using the Pichia pastoris codon preference 3-OST-1 gene (SEQ ID NO.5) as a template. The operation steps were the same as those of the above-mentioned Escherichia coli, but the primers were different; the specific primer sequences are as follows:

[0100] F2': 5'-AGAAAGAGAGGCTGAAGCTAGACCAGCTGAATTGGGTCAAC-3'; SEQ ID NO.14;

[0101] R2': 5'-CGTGGGAGTAGTGCTCTCTCTATCAAAAAAATGAACTTCGTTTTCA GCAGC-3'; SEQ ID NO.15;

[0102] F3': 5'-GAGGAGCACTACTCCCACGG-3'; SEQ ID NO.16;

[0103] R3': 5'-CAAACTCAATGATGATGATGATGATGATGCCAGTCGAAGGTACGAC-3'; SEQ ID NO. 17;

[0104] F4': 5'-ATGCAGAAGCACAAGCCATACC-3'; SEQ ID NO.18;

[0105] R4': 5'-TATGGCTTGTGCTTCTGCATTCTGTTGTAAAAAACTTGAGTGTAGTCA GACAAAAC-3'; SEQ ID NO. 19.

[0106] The plasmid construction, induced expression and activity testing methods are the same as those in Example 2.

[0107] The results are as follows Figure 5 and Figure 6 As shown. The enzyme catalytic activity of wild-type (WT) 3-OST-1 expressed by E. coli OrigamiB (DE3) was 15.605U / L; the enzyme catalytic activity of 3-OST-1 (W72R) expressed by E. coli OrigamiB (DE3) after the amino acid at position 72 was mutated from tryptophan to arginine was 26.398U / L, which was 69% higher than that before the mutation; the enzyme catalytic activity of 3-OST-1 (H144R) expressed by E. coli OrigamiB (DE3) after the amino acid at position 144 was mutated from histidine to arginine was 17.214U / L, which was 10% higher than that before the mutation; the enzyme catalytic activity of 3-OST-1 (W72R / H144R) expressed by E. coli OrigamiB (DE3) after the amino acid at position 72 was mutated from tryptophan to arginine and the amino acid at position 144 was mutated from histidine to arginine was 34.411U / L, which was 121% higher than that before the mutation. 1L of bacterial solution expressing 3-OST-1(W72R / H144R) enzyme can be purified and concentrated to obtain 2mL 0.3mg / mL pure enzyme solution. The specific enzyme activity and sulfotransfer rate of 3-OST-1(W72R / H144R) pure enzyme were measured according to the method in Example 1. The specific enzyme activity was 2.40U / mg, which was 43% higher than that of the wild type, and the sulfotransfer rate was 23.61pmol / μg, which was 82% higher than that of the wild type.

[0108] The enzyme catalytic activity of wild-type 3-OST-1 (3-OST-1(WT)) expressed by Pichia pastoris GS115 was 66.773U / L; the enzyme catalytic activity of 3-OST-1 (W72R) expressed by Pichia pastoris GS115 after the amino acid at position 72 was mutated from tryptophan to arginine was 70.271U / L, which was 5% higher than that before the mutation; the enzyme catalytic activity of 3-OST-1 (H144R) expressed by Pichia pastoris GS115 after the amino acid at position 144 was mutated from histidine to arginine was 68.857U / L, which was 5% higher than that before the mutation. The enzyme activity of wild-type 3-OST-1 (MBP-3-OST-1 (WT)) with N-terminal fusion MBP tag expressed by Pichia pastoris GS115 was 75.408U / L, which was 13% higher than that of the wild-type before the tag was fused; the enzyme activity of 3-OST-1 after the N-terminal fusion MBP protein was expressed by Pichia pastoris GS115 and the amino acid at position 72 was mutated from tryptophan to arginine was 88.509U / L, which was 17% higher than that of the wild-type with fusion-promoting tag and 33% higher than that of the wild-type without fusion-promoting tag.

[0109] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A heparin 3-O-sulfotransferase 1 mutant, characterized in that: The solubility-promoting tag MBP is fused to the N-terminus of heparin 3-O-sulfotransferase 1, and then a heparin 3-O-sulfotransferase 1 mutant is obtained by molecular modification; the molecular modification includes any one of (1) to (3): (1) The amino acid 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine; (2) mutating amino acid 144 of heparin 3-O-sulfotransferase 1 from histidine to arginine; (3) mutating the amino acid at position 72 of heparin 3-O-sulfotransferase 1 from tryptophan to arginine, and mutating the amino acid at position 144 from histidine to arginine; The amino acid sequence of the heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.2; the amino acid sequence of the MBP is shown in SEQ ID NO.

7.

2. A gene encoding the heparin 3-O-sulfotransferase 1 mutant according to claim 1.

3. A plasmid containing the gene according to claim 2.

4. A genetically engineered bacterium expressing the heparin 3-O-sulfotransferase 1 mutant according to claim 1.

5. A method for producing heparin 3-O-sulfotransferase 1, characterized in that: The genetically engineered bacteria according to claim 4 are used as production strains to induce fermentation to produce heparin 3-O-sulfotransferase 1.

6. A method for improving the catalytic activity of heparin 3-O-sulfotransferase 1, characterized in that: First, a solubility-promoting tag MBP is fused to the N-terminus of heparin 3-O-sulfotransferase 1, and then a heparin 3-O-sulfotransferase 1 mutant is obtained by molecular modification; the molecular modification includes any one of (1) to (3): (1) The amino acid 72 of heparin 3-O-sulfotransferase 1 was mutated from tryptophan to arginine; (2) mutating amino acid 144 of heparin 3-O-sulfotransferase 1 from histidine to arginine; (3) mutating the amino acid at position 72 of heparin 3-O-sulfotransferase 1 from tryptophan to arginine, and mutating the amino acid at position 144 from histidine to arginine; The amino acid sequence of the heparin 3-O-sulfotransferase 1 is shown in SEQ ID NO.2; the amino acid sequence of the MBP is shown in SEQ ID NO.

7.

7. Use of the heparin 3-O-sulfotransferase 1 mutant according to claim 1 in improving the activity of heparin 3-O-sulfotransferase 1.

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