Heparin 3-O-sulfuryl transferase mutant with high enzyme activity and high thermal stability as well as coding gene and application of heparin 3-O-sulfuryl transferase mutant
By performing site-directed mutagenesis and structural optimization on the 3OST truncated form of the Red Junglefowl, a 3-O-sulfate transferase mutant SM1 with high enzyme activity and high thermostability was constructed, which solved the problem of insufficient enzyme activity and stability in the existing technology and improved the efficiency and feasibility of heparin synthesis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG UNIV
- Filing Date
- 2026-02-28
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, 3-O-sulfate transferases exhibit low catalytic efficiency and poor protein stability in heterologous expression systems, and are particularly prone to inactivation at reaction temperatures, making it difficult to meet the requirements of high activity, high thermal stability, and high expression in the chemical enzymatic synthesis of heparin.
By selecting the truncated form Gg-3OST-1 from the genus *Gnaphalium rubrum* and performing site-directed mutagenesis at 24 amino acid sites, a 3-O-sulfate transferase mutant SM1 with high enzyme activity and high thermostability was constructed. The N-terminal signal peptide was removed, and its structure was optimized by combining glycosylation engineering and the PROSS algorithm.
It achieved a synergistic improvement in catalytic activity and thermal stability, with total enzyme activity increased by 1.5 times and thermal stability significantly improved. After being incubated at 37°C for 8 days, it could still maintain 50% of the initial activity, which significantly improved the efficiency and feasibility of heparin synthesis by chemical enzymatic method.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioenzyme technology, specifically to a heparin 3-O-sulfate transferase mutant with high total enzyme activity and high thermal stability derived from the genus *Gallus gallu*, its encoding gene, and its applications. Background Technology
[0002] Heparin (HP), a highly sulfated glycosaminoglycan, is composed of repeating disaccharide units formed by D-β-glucuronic acid (or L-α-iduronic acid) and N-acetylglucosamine. Heparin is an indispensable anticoagulant globally, serving as a cornerstone drug for clinical anticoagulation and antithrombotic therapy. It is widely used in acute coronary syndrome, deep vein thrombosis, pulmonary embolism prevention and treatment, hemodialysis, and extracorporeal circulation during cardiac surgery. Furthermore, the discovery of its non-anticoagulant activities, such as anti-inflammatory, antiviral, and tumor metastasis inhibition, has further expanded its clinical applications. In 2026, the global heparin market exceeded US$8 billion. China, as a major producer and consumer of heparin drugs, has a formulation market size of RMB 28 billion, with low molecular weight heparin (LMWH) accounting for over 70%. Under the normalization of centralized procurement, mainstream products such as enoxaparin and dalteparin are accelerating domestic substitution, driving the industry towards higher value-added products.
[0003] The core pharmacological activity of heparin, particularly its ability to bind to and activate thrombin III (AT), is primarily determined by the key 3-O-sulfate group in the heparin pentasaccharide sequence. However, traditional production methods that extract heparin from animal tissues such as porcine intestinal mucosa suffer from inherent drawbacks, including strong dependence on raw materials, complex processes, batch-to-batch quality inconsistencies, and potential risks of viral contamination and immunogenicity (such as the 2008 heparin contamination incident). Therefore, developing synthetic heparin with a defined structure, high purity, and safety has become a strategic direction in the field of glycopharmaceuticals.
[0004] Among various synthetic pathways, the chemoenzymatic synthesis strategy is considered the optimal route for achieving large-scale production of heparin oligosaccharides and analogues with uniform structure and controllable activity due to its ability to construct complex sulfated sugar chain structures with high stereoselectivity and stepwise steps. The core of this strategy is the use of a series of sulfate transferases with specific regioselectivity. 3-O-sulfate transferase-1 (3-OST-1), as the final modifying enzyme catalyzing the generation of key anticoagulant sites, directly determines the potency and quality of the final product, thus becoming the "rate-limiting enzyme" and a key bottleneck in the entire technology chain.
[0005] To overcome the problems of low catalytic efficiency, poor protein stability (especially easy inactivation at reaction temperature), and insufficient soluble expression levels that are common in natural 3-OST-1 heterologous expression systems, researchers at home and abroad have tried a variety of protein engineering strategies, but all of them have significant limitations and cannot meet the triple requirements of "high activity, high thermal stability, and high expression" for enzyme preparations in chemical enzymatic synthesis. Summary of the Invention
[0006] To address the problems existing in the prior art, this invention provides a heparin 3-O-sulfate transferase mutant with high enzyme activity and high thermostability, along with its encoding gene and applications. Specifically, by exploring 3OST from other sources and removing the N-terminal signal peptide to construct corresponding 3OST truncated variants, a 3OST truncated variant from the genus *Gnaphalium affine* (hereinafter referred to as Gg-3OST-1) was successfully obtained. Furthermore, protein engineering was performed on Gg-3OST-1 to improve its total enzyme activity and stability.
[0007] Terminology Explanation
[0008] GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP: The full Chinese name is p-nitrophenyl-glucuronic acid-N-sulfate-6-O-sulfate glucosamine-2-O-sulfate iduronic acid-N-sulfate-6-O-sulfate glucosamine-glucuronic acid-N-sulfate-6-O-sulfate glucosamine. It is a substrate of 3-O-sulfate transferase, which catalyzes the formation of GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S-GlcA-pNP.
[0009] PAPS: The full Chinese name is 3′-adenosine phosphate-5′-phosphoryl sulfate, which is a sulfate donor.
[0010] The technical solution of this invention is as follows:
[0011] In a first aspect, the present invention provides a 3-O-sulfate transferase mutant SM1, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1;
[0012] The 3-O-sulfotransferase mutant SM1 was first developed from wild-type chicken 3-O-sulfotransferase (Gg-3OST-WT), by selecting a fragment from alanine (A23) to serine (S320) at position 23 in the complete amino acid sequence as a chicken 3-O-sulfotransferase truncated variant (Gg-3OST-1); then, based on the chicken 3-O-sulfotransferase truncated variant (Gg-3OST-1), site-directed mutations were performed at 24 amino acid sites.
[0013] According to a preferred embodiment of the present invention, the NCBI accession number of the wild-type chicken 3-O-sulfotransferase (Gg-3OST-WT) is XP_040525872.1.
[0014] According to a preferred embodiment of the present invention, the site-directed mutations at the 24 amino acid sites are: S22N, Q23N, R38Q, H81N, R84K, I90R, Q124P, S125A, R127K, K166R, V172T, N178Q, H197Q, K205Q, K208R, K222G, N228S, A229P, S230N, A259P, T265P, H272R, Y276R, and S295C. These site-directed mutations begin with the first position (corresponding to A23 in the wild-type complete sequence) of the chicken 3-O-sulfate transferase truncated variant (Gg-3OST-1), with subsequent positions numbered sequentially, clearly defining the relative position of the mutation site within the functional domain.
[0015] Compared with the chicken-derived 3-O-sulfate transferase truncated variant, the SM1 mutant of 3-O-sulfate transferase has the following mutations: serine at position 22 is mutated to asparagine; glutamine at position 23 is mutated to asparagine; arginine at position 38 is mutated to glutamine; histidine at position 81 is mutated to asparagine; arginine at position 84 is mutated to lysine; isoleucine at position 90 is mutated to arginine; glutamine at position 124 is mutated to proline; serine at position 125 is mutated to alanine; arginine at position 127 is mutated to lysine; lysine at position 166 is mutated to arginine; and valine at position 172 is mutated to threonine. The following mutations were made: asparagine at position 178 was replaced by glutamine; histidine at position 197 was replaced by glutamine; lysine at position 205 was replaced by glutamine; lysine at position 208 was replaced by arginine; lysine at position 222 was replaced by glycine; asparagine at position 228 was replaced by serine; alanine at position 229 was replaced by proline; serine at position 230 was replaced by asparagine; alanine at position 259 was replaced by proline; threonine at position 265 was replaced by proline; histidine at position 272 was replaced by arginine; tyrosine at position 276 was replaced by arginine; and serine at position 295 was replaced by cysteine.
[0016] The 3-O-sulfotransferase mutant SM1 provided in this invention is designed based on a preferred wild-type chicken 3-O-sulfotransferase (Gg-3OST-WT). To balance prokaryotic expression efficiency and enzymatic functional integrity, the wild-type Gg-3OST-WT was structurally optimized: the non-catalytic signal peptide sequence at the N-terminus was removed, and a fragment from alanine (A23) to serine (S320) at position 23 of the complete amino acid sequence was selected as the "chicken 3-O-sulfotransferase truncated variant" (i.e., the A23-S320 fragment), hereinafter referred to as Gg-3OST-1. This fragment fully contains the core catalytic domain and substrate-specific binding site of 3-OST, ensuring the key function of the enzymatic reaction. Then, based on the chicken 3-O-sulfotransferase truncated variant (Gg-3OST-1), site-directed mutagenesis was performed at 24 amino acid sites to obtain higher enzyme activity and higher thermostability.
[0017] A second aspect of the invention provides a recombinant vector containing the coding gene of the 3-O-sulfate transferase mutant SM1. The recombinant vector is not particularly limited in its originating vector and can be any vector known in the art, as long as it can replicate in the host. For example, the vector includes, but is not limited to, plasmids and bacteriophages. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself.
[0018] According to a preferred embodiment of the present invention, the recombinant vector is obtained by ligating the coding gene of the 3-O-sulfotransferase mutant SM1 to a plasmid.
[0019] More preferably, the plasmid is a pMAL-c5x expression vector (containing an MBP tag to promote protein folding).
[0020] A third aspect of the invention provides recombinant cells containing the encoding gene of the 3-O-sulfate transferase mutant SM1.
[0021] According to a preferred embodiment of the present invention, the recombinant cell is obtained by transforming the encoding gene of the 3-O-sulfate transferase mutant SM1 into a host cell. The term "host cell" has the meaning commonly understood in the art; it refers to a host cell capable of introducing the encoding gene of the mutant of the present invention, and is called a recombinant cell after introduction. The strain of the present invention can be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, and more preferably an *Escherichia coli* OrigamiB(DE3) containing the molecular chaperone pGro7 helper vector, hereinafter referred to as OrigamiB(DE3)-pGro7.
[0022] In a fourth aspect, the present invention provides a method for producing a 3-O-sulfate transferase mutant SM1 by fermentation, the method comprising: culturing the above-mentioned recombinant cells, and then isolating and obtaining the 3-O-sulfate transferase mutant SM1.
[0023] According to a preferred embodiment of the present invention, the specific steps of the method for producing the 3-O-sulfate transferase mutant SM1 by fermentation are as follows: recombinant cells containing the encoding gene of the 3-O-sulfate transferase mutant SM1 are cultured in LB liquid medium until the OD600 is 0.6-0.8, and IPTG is added to induce expression for 16-18 h; the cells are collected, sonicated, centrifuged and filtered, and purified by MBP tagging to obtain the 3-O-sulfate transferase mutant SM1.
[0024] A fifth aspect of the present invention provides the use of the above-described 3-O-sulfatyltransferase mutant SM1 in the synthesis of 3-O-sulfated heparin oligosaccharides or polysaccharides.
[0025] According to a preferred embodiment of the present invention, the application uses PAPS as a donor and heparin oligosaccharides and polysaccharides having a structure containing the GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP as the acceptor substrate as the initiating acceptor to catalyze a reaction to generate 3-O-sulfated heparin oligosaccharides or polysaccharides with the structure GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S--GlcA-pNP.
[0026] Experimental procedures not described in detail in this invention can be performed according to conventional experimental procedures in this technical field.
[0027] Beneficial effects
[0028] 1. This invention provides a 3-O-sulfotransferase mutant SM1 with high enzyme activity, high catalytic activity, and high thermostability, exhibiting significantly superior performance in the enzymatic synthesis of heparin-like glycosaminoglycans. Performance measurements show that the 3-O-sulfotransferase mutant SM1 achieves a synergistic improvement in catalytic activity and thermostability, with its total enzyme activity increasing by 1.5 times compared to wild-type chicken 3-O-sulfotransferase (Gg-3OST-WT). More notably, its thermostability is fundamentally improved: wild-type chicken 3-O-sulfotransferase (Gg-3OST-WT) typically exhibits rapid activity decay within 24 hours at 37°C, while the 3-O-sulfotransferase mutant SM1 retains approximately 50% of its initial activity after incubation under the same conditions for 8 days, demonstrating excellent operational stability. The total 3-O-sulfate synthesis efficiency per liter of bacteria over 24 hours is increased from 2.736 mM for the wild type to 6.895 mM. In other words, the 3-O-sulfate transferase mutant SM1 provided by this invention effectively overcomes the key bottlenecks in the industrial application of natural enzymes, significantly improving the efficiency and feasibility of chemical enzymatic synthesis of heparin and its analogues. Its high thermal stability broadens the operating window of the enzymatic reaction, laying a core foundation for developing a more efficient and economical biomimetic synthesis process for heparin, and is of great significance for promoting the advancement of glycosaminoglycan drug production technology.
[0029] 2. Compared with wild-type chicken 3-O-sulfotransferase (Gg-3OST-WT), the 3-O-sulfotransferase mutant SM1 provided in this invention is obtained through a synergistic superposition mutation strategy. Specifically, the 3-O-sulfotransferase mutant SM1 is constructed by combining the advantageous mutant M7 obtained through glycosylation engineering with the stability-enhancing mutant D5 designed using the PROSS algorithm. The 3-O-sulfotransferase mutant SM1 contains mutations at 24 key amino acid sites, exhibiting clear catalytic specificity. It can efficiently catalyze the 3-O-sulfation modification of the heparin characteristic hexasaccharide substrate GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP using 3'-adenosine-5'-phosphate sulfate (PAPS) as the sulfate donor. Attached Figure Description
[0030] Figure 1 Comparison of total enzyme activities of 3OST from ten different species expressed in Escherichia coli;
[0031] In the figure: the horizontal axis represents the species names of 3OST, and the vertical axis represents the total enzyme activity.
[0032] Figure 2 A comparison of the half-lives of 3OST from ten different species expressed in Escherichia coli;
[0033] In the figure: the horizontal axis represents the species name of 3OST, and the vertical axis represents enzyme activity.
[0034] Figure 3 Comparison of total enzyme activity among seven Gg-3OST-1 mutants obtained by glycosylation site mutation;
[0035] In the figure: the horizontal axis represents the Gg-3OST-1 mutant, and the vertical axis represents the total enzyme activity.
[0036] Figure 4 Comparison of the half-life of the Gg-3OST-M7 mutant obtained by glycosylation site mutation with that of WT;
[0037] In the graph: the horizontal axis represents storage time, and the vertical axis represents conversion rate.
[0038] Figure 5 Comparison of total enzyme activity among seven Gg-3OST-1 mutants calculated by PROSS;
[0039] In the figure: the horizontal axis represents the Gg-3OST-1 mutant, and the vertical axis represents the total enzyme activity.
[0040] Figure 6 Comparison of the half-life of the Gg-3OST-D5 mutant and WT as calculated by PROSS;
[0041] In the graph: the horizontal axis represents storage time, and the vertical axis represents conversion rate.
[0042] Figure 7 Comparison of total enzyme activity and half-life between Gg-3OST-M7 mutant, Gg-3OST-D5 mutant, stacked mutant SM1 and wild-type chicken 3-O-sulfotransferase (WT);
[0043] In the figure: the horizontal axis represents half-life, and the vertical axis represents total enzyme activity. Detailed Implementation
[0044] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental operations involved in the embodiments are all conventional operations in the art. The materials, reagents, etc. used in the embodiments can all be obtained commercially.
[0045] In the following examples, all 3-O-sulfate transferases and their mutants were recombinantly expressed and purified in E. coli using the following methods:
[0046] The amino acid sequences of all 3-O-sulfate transferases and their mutants in this invention were optimized by E. coli codons, and then synthesized or mutated by Nanjing Genscript Co., Ltd. according to the sequence information. They were then cloned into the pMAL-c5x vector and chemically transformed into OrigamiB(DE3)-Pgro7 competent cells. The cells were cultured on LB solid medium containing carbenicillin (50 μg / mL) and chloramphenicol (35 μg / mL) for 12 h, and transformants were screened (a negative control experiment was performed at the same time) to obtain positive transformants.
[0047] Single colonies of 3-O-sulfate transferase and its mutant positive transformants were picked and activated in 20 mL of sterile LB broth (containing 50 μg / mL carbenicillin and 35 μg / mL chloramphenicol) at 37°C and 225 rpm. The activated bacterial culture was then inoculated at a 1% inoculation rate into 100 mL of LB broth (containing 50 μg / mL carbenicillin and 35 μg / mL chloramphenicol) and cultured with shaking at 37°C and 225 rpm for 4 hours until OD reached. 600 Approximately 0.8 mg / L was added to a final concentration of 0.2 mM IPTG, and expression was induced at 22℃ and 225 rpm for 16–18 h. The bacterial cells were collected by centrifugation and resuspended in 1×PBS buffer. The cells were then sonicated on ice (3 s on, 5 s off, 33% amplitude, 1500 kJ energy, 4℃) for 40 s. The lysed cells were centrifuged at 12000 rpm for 20 min (4℃), and the supernatant was filtered through a 0.22 μm filter. Purification was performed using an MBP column. After loading, the cells were washed with 1×PBS buffer, and finally eluted with 1×PBS buffer containing 20 mM maltose to obtain the target protein. The purified protein was stored in 20% glycerol, aliquoted, and stored at -80℃.
[0048] The purified recombinant protein was identified by polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration of recombinant and mutant proteins was determined using a BCA protein assay kit (Beyotime P0011).
[0049] In the following examples, the activity and stability of all 3-O-sulfate transferases and their mutants were determined using the following methods:
[0050] 1. Determination of sulfate transfer activity
[0051] The hexasaccharide GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP (6mer-6s) was used as the acceptor substrate, and PAPS was used as the donor substrate. The reaction system is shown in Table 1. The reaction system was placed in a water bath at 37°C for 1 h, and then heated with boiling water for 5 min to inactivate the test enzyme (3-O-sulfotransferase and its mutant), thereby terminating the reaction. The reaction solution was filtered through a 0.22 μm filter membrane and then analyzed by HPLC according to the method described in Table 2. The pNP group of the monosaccharide acceptor showed specific absorption at a UV detection wavelength of 310 nm, and the mobile phase flow rate was 0.5 mL / min.
[0052] Table 1. Reaction systems for determining the sulfate transfer activity of 3-O-sulfatyltransferase and its mutants.
[0053]
[0054] Table 2. HPLC analytical methods used for detecting heparin oligosaccharides
[0055]
[0056] 2. Stability determination of sulfate transfer activity
[0057] The hexasaccharide GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP (6mer-6s) was used as the acceptor substrate, and PAPS was used as the donor substrate. The reaction system is shown in Table 1. First, the test enzyme (3-O-sulfotransferase and its mutant) was incubated at 37℃, and samples were taken every 24 hours to detect enzyme activity. The reaction was then carried out in a water bath at 37℃ for 1 hour, followed by boiling in water for 5 minutes to inactivate the enzyme and terminate the reaction. The reaction solution was filtered through a 0.22 μm filter and analyzed by HPLC according to the method described in Table 2. The pNP group of the monosaccharide acceptor showed specific absorption at a UV detection wavelength of 310 nm, and the mobile phase flow rate was 0.5 mL / min.
[0058] Example 1: Screening for the optimal species-derived 3-O-sulfate transferase cutoff from wild-type 3-O-sulfate transferases from ten different species.
[0059] Using the human 3-O-sulfotransferase 1 (Hs-3OST-1, NCBI accession number AAH57803.1), which has been confirmed to have heparin anticoagulant activity, as the probe sequence, we systematically mined homologous genes in 16 model organisms using the NCBI homology search tool, aiming to expand the research framework of the 3OST enzyme family and screen artificially evolved candidate sequences.
[0060] Based on the principle of evolutionary representativeness, 10 3-O-sulfotransferase homologs from different species were screened from the retrieved homologous sequences. These homologs cover higher mammals (Homo sapiens), birds (Gallus gallus), amphibians (Xenopus laevis), fish (Danio rerio zebrafish, Oryzias latipes, Gasterosteus aculeatus), arthropods (Apis mellifera), cnidarians (Caenorhabditis elegans), invertebrates (Branchiostoma floridae), and basic multicellular animals (Strongylocentrotus purpuratus), covering key evolutionary nodes from lower to higher organisms.
[0061] Among them, the NCBI accession number of Gg-3OST (chicken source) is XP_040525872.1;
[0062] The NCBI accession number for XI-3OST (from the African clawed frog) is NP_001083522.1;
[0063] The NCBI accession number for OI-3OST (medaka fish source) is XP_004073075.1;
[0064] The NCBI accession number for Ga-3OST (spinyfish source) is XP_040031098.1;
[0065] The NCBI accession number for Dr-3OST (zebrafish source) is XP_005173108.1;
[0066] The NCBI registration number for Am-3OST (Italian bee source) is XP_006569304.1;
[0067] The NCBI registration number for Bf-3OST (Wenchang Fish Source) is XP_035666584.1;
[0068] The NCBI accession number for Sp-3OST (purple sea urchin source) is XP_799088.1;
[0069] The NCBI accession number for Ce-3OST (Caenorhabditis elegans) is ABU48855.1.
[0070] Among them, compared with human 3-O-sulfotransferase, human 3-O-sulfotransferase 1 is a truncated form with 22 amino acids. Therefore, the same method was used to truncate wild-type 3-O-sulfotransferases from the other 9 different species, resulting in Gg-3OST-1 (chicken 3-O-sulfotransferase truncated form), XI-3OST-1, OI-3OST-1, Ga-3OST-1, Dr-3OST-1, Am-3OST-1, Bf-3OST-5, Sp-3OST-1 and Ce-3OST-1.
[0071] Then, the 3OST-1 genes (coding sequences) from 10 different sources, including Hs-3OST-1, Gg-3OST-1, XI-3OST-1, OI-3OST-1, Ga-3OST-1, Dr-3OST-1, Am-3OST-1, Bf-3OST-5, Sp-3OST-1, and Ce-3OST-1, were cloned into the pMal-C5X expression vector and transformed into Escherichia coli OrigamiB(DE3)-pGro7 host cells. Following the aforementioned recombinant expression and purification methods, heterologous expression and MBP affinity chromatography were performed to obtain recombinant Hs-3OST-1, Gg-3OST-1, XI-3OST-1, OI-3OST-1, Ga-3OST-1, Dr-3OST-1, Am-3OST-1, Bf-3OST-5, Sp-3OST-1, and Ce-3OST-1 proteins.
[0072] Following the aforementioned methods for determining sulfate transfer activity and stability, the total enzyme activity and stability of 10 recombinant proteins from different sources were determined. The results are as follows: Figure 1 and Figure 2 As shown.
[0073] Depend on Figure 1 and Figure 2 It is known that the chicken-derived Gg-3OST-1 selected from the higher animal cluster has the best performance and the highest total enzyme activity, but its half-life of only 24 hours severely limits its potential for practical application. This characteristic also makes it an ideal starting template for breaking through the constraints of natural evolution and obtaining a dual-superior enzyme of "high activity and high stability" through protein engineering.
[0074] Example 2: Rational design of mutations at glycosylation sites to obtain the Gg-3OST-1 mutant
[0075] Using the ESPript tool, homologous sequences of 3OST-1 from various species were compared and analyzed. Combined with the evolutionary pattern of glycosylation sites (from 2 sites in fish → up to 9 sites in amphibians / reptiles → 4-5 sites in higher mammals – a “simple → complex → simple” evolutionary trajectory), it was determined that chicken-derived Gg-3OST-1 contains 5 glycosylation sites, 3 of which are non-conserved sites in low-glycosylation species (corresponding to truncated regions Q124, S125, N178, N228, A229, and S230). Based on the hypothesis that “low-glycosylated species compensate for glycosylation loss through amino acid mutations,” the asparagine (Asn) residues at the above 3 non-conserved glycosylation sites were mutated into conserved non-glycosylated residues in low-glycosylated 3OST-1 (such as glutamine Gln, proline Pro, and alanine Ala), which were identified as the core targets for subsequent glycosylation mutation research.
[0076] Based on the identified non-conserved glycosylation targets, specific primers were designed, and a series of glycosylation mutants were constructed using PCR-mediated site-directed mutagenesis, based on the chicken 3-O-sulfotransferase truncated variant. These mutants are:
[0077] Gg-3OST-1-M1: Q124P / S125A (the truncated form has glutamine at position 124 mutated to proline and serine at position 125 mutated to alanine), which is a mutation of 2 amino acids compared to the wild-type Gg-3OST-1 (truncated form);
[0078] Gg-3OST-1-M2:N178Q (the truncated form has an asparagine mutation at position 178 to glutamine), which is a mutation of one amino acid compared to the wild type;
[0079] Gg-3OST-1-M3: N228S / A229P / S230N (the truncated form has three amino acid mutations: asparagine at position 228 is replaced by serine, alanine at position 229 is replaced by proline, and serine at position 230 is replaced by asparagine), compared to the wild type.
[0080] Gg-3OST-1-M4: Q124P / S125A / 228S / A229P / S230N (the truncated form has glutamine at position 124 mutated to proline, serine at position 125 mutated to alanine, asparagine at position 228 mutated to serine, alanine at position 229 mutated to proline, and serine at position 230 mutated to asparagine), which is a mutation in 5 amino acids compared to the wild type;
[0081] Gg-3OST-1-M5: N178Q / N228S / A229P / S230N (the truncated form has four amino acid mutations compared to the wild type: asparagine at position 178 is replaced by glutamine, asparagine at position 228 is replaced by serine, alanine at position 229 is replaced by proline, and serine at position 230 is replaced by asparagine).
[0082] Gg-3OST-1-M6: Q124P / S125A / N178Q (the truncated form has glutamine at position 124 mutated to proline, serine at position 125 mutated to alanine, and asparagine at position 178 mutated to glutamine), which is a mutation of 3 amino acids compared to the wild type;
[0083] 3OST-1-M7: Q124P / S125A / N178Q / N228S / A229P / S230N (the truncated form has 6 amino acid mutations compared to the wild type: glutamine at position 124 is replaced by proline, serine at position 125 is replaced by alanine, asparagine at position 178 is replaced by glutamine, asparagine at position 228 is replaced by serine, alanine at position 229 is replaced by proline, and serine at position 230 is replaced by asparagine).
[0084] The encoding genes of Gg-3OST-1-M1~7 were cloned into the pMAL-c5x expression vector. After the mutant plasmid was verified to be correct by sequencing, it was transformed into Escherichia coli OrigamiB (DE3)-pGro7 host cells. Heterologous expression and MBP affinity chromatography were performed according to the aforementioned recombinant expression and purification methods to obtain recombinant Gg-3OST-1-M1, Gg-3OST-1-M2, Gg-3OST-1-M3, Gg-3OST-1-M4, Gg-3OST-1-M5, Gg-3OST-1-M6 and Gg-3OST-1-M7 proteins, respectively.
[0085] Following the aforementioned methods for determining sulfate transfer activity and stability, the total enzyme activity of seven mutants (M1-7) and wild-type chicken 3-O-sulfotransferase (WT) was measured, and the stability of Gg-3OST-1-M7 and WT was determined. The results are as follows: Figure 3 and Figure 4 As shown.
[0086] Depend on Figure 3 and Figure 4 It can be seen that the three-point mutant M7 exhibits the best overall performance, with a 1.6-fold increase in total enzyme activity, a 4.5-fold increase in half-life, and a significant increase in the expression of soluble protein without loss of natural catalytic activity. Therefore, M7 was identified as the core engineering template after glycosylation optimization.
[0087] Example 3: PROSS algorithm calculation yielded a 3-O-sulfate transferase mutant with enhanced thermal stability.
[0088] The three-dimensional structure of wild-type chicken 3-O-sulfotransferase was obtained (predicted by AlphaFold2). Based on homologous sequence evolutionary conservation analysis and using the PROSS algorithm, stability optimization design was performed on amino acid sites far from the active site. The PROSS algorithm calculated and designed seven Gg-3OST-1 mutants that enhance stability. The specific mutation details of each mutant (based on the site numbering of the chicken 3-O-sulfotransferase truncated variant Gg-3OST-1) are as follows:
[0089] Gg-3OST-1-D1: Compared to Gg-3OST-1, six amino acids have been mutated, specifically: H80R, R84K, V172T, K205Q, K208R, and K222G;
[0090] Gg-3OST-1-D2: Compared to Gg-3OST-1, eight amino acids are mutated, specifically: S22N, Q23N, R38Q, H80R, R84K, V172T, K205Q, and K208R.
[0091] Gg-3OST-1-D3: Compared to Gg-3OST-1, 12 amino acids are mutated, specifically: S22N, Q23N, R38Q, H80R, R84K, I90R, V172T, N178Q, H197Q, K205Q, K208R, K222G;
[0092] Gg-3OST-1-D4: Compared to Gg-3OST-1, 14 amino acids are mutated, specifically: S22N, Q23N, R38Q, H80N, R84K, I90R, V172T, N178Q, H197Q, K205Q, K208R, K222G, N228S, A229P;
[0093] Gg-3OST-1-D5: Compared to Gg-3OST-1, 14 amino acids are mutated, specifically: S22N, Q23N, R38Q, H80N, R84K, I90R, V172T, N178Q, H197Q, K205Q, K208R, K222G, N228S, S230N;
[0094] Gg-3OST-1-D6: Compared to Gg-3OST-1, 20 amino acids are mutated, specifically: S22N, Q23N, R38Q, H80N, R84K, I90R, Q124P, S125A, R127K, V172T, N178Q, H197Q, K205Q, K208R, K222G, N228S, A229P, S230N, A259P, T265P;
[0095] Gg-3OST-1-D7: Compared to Gg-3OST-1, 38 amino acids are mutated, specifically: T8A, S9R, S12A, S22N, Q23N, R38Q, E70K, N71E, H80N, R84K, I90R, Q124P, S125A, R127K, K166R, E169Q, V172T, N178Q, H197Q, K205Q, K208R, K222G, N228S, A229P, S230N, A259P, T265P, H272R, Y276R, and S295C (the remaining mutation sites correspond to characteristic residues in the sequence alignment region).
[0096] The encoding genes of Gg-3OST-1-D1~7 were cloned into the pMAL-c5x expression vector. After the mutant plasmid was verified to be correct by sequencing, it was transformed into Escherichia coli OrigamiB (DE3)-pGro7 host cells. Heterologous expression and MBP affinity chromatography were performed according to the aforementioned recombinant expression and purification methods to obtain recombinant Gg-3OST-1-D1, Gg-3OST-1-D2, Gg-3OST-1-D3, Gg-3OST-1-D4, Gg-3OST-1-D5, Gg-3OST-1-D6 and Gg-3OST-1-D7 proteins, respectively.
[0097] Following the aforementioned methods for determining sulfate transfer activity and stability, the total enzyme activity of seven mutants (D1-7) and wild-type chicken 3-O-sulfotransferase (WT) was measured, and the stability of Gg-3OST-1-D5 and WT was determined. The results are as follows: Figure 5 and Figure 6 As shown.
[0098] Depend on Figure 5 and Figure 6 It can be seen that Gg-3OST-1-D5 exhibits the best activity and stability while maintaining a high soluble expression level. Therefore, it has been identified as the core candidate mutant after PROSS modification for subsequent functional studies and engineering applications.
[0099] Example 4: The mutant 3OST-1-SM1 was obtained by superimposing the two methods.
[0100] The mutant Gg-3OST-1-M7 (glycosylation site optimized mutation) obtained based on rational glycosylation design in Example 2 was integrated with other mutation sites except glycosylation sites in the mutant Gg-3OST-1-D5 obtained based on PROSS engineering design in Example 3 to construct the stacked mutant SM1.
[0101] The specific mutation sites of the stacked mutant SM1 (based on the site numbering of the chicken 3-O-sulfatyltransferase truncated variant) are: glycosylation mutations at the integration site of M7, and mutations in D5 excluding the glycosylation site, totaling 24 amino acid sites. Specifically, these are: S22N, Q23N, R38Q, H81N, R84K, I90R, Q124P, S125A, R127K, K166R, V172T, N178Q, H197Q, K205Q, K208R, K222G, N228S, A229P, S230N, A259P, T265P, H272R, Y276R, and S295C.
[0102] The coding gene of the stacked mutant SM1 was cloned into the pMAL-c5x expression vector. After the mutant plasmid was verified to be correct by sequencing, it was transformed into Escherichia coli OrigamiB (DE3)-pGro7 host cells. The recombinant SM1 protein was obtained by heterologous expression and MBP affinity chromatography according to the aforementioned recombinant expression and purification methods.
[0103] Following the aforementioned methods for determining sulfate transfer activity and stability, the total enzyme activity and stability of Gg-3OST-M7 mutant, Gg-3OST-D5 mutant, stacked mutant SM1, and wild-type chicken 3-O-sulfotransferase (WT) were determined. The results are as follows: Figure 7 As shown.
[0104] Depend on Figure 7 It was found that the total enzyme activity of the stacked mutant SM1 was 1.5 times that of the wild type, and the half-life was extended to 8 days (8 times that of the wild type). This indicates that 3OST-1-SM1 has extremely high stability compared to wild-type 3OST-1, which suggests its high application value and potential in heparin synthesis.
[0105] The embodiments described above are merely preferred implementations of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A 3-O-sulfotransferase mutant SM1, characterized in that, The amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence encoding the gene is shown in SEQ ID NO.
1.
2. A recombinant vector containing the encoding gene of the 3-O-sulfate transferase mutant SM1 as described in claim 1.
3. The recombinant vector as described in claim 1, characterized in that, The recombinant vector was obtained by ligating the coding gene of the 3-O-sulfate transferase mutant SM1 to a plasmid; the plasmid was the pMAL-c5x expression vector.
4. A recombinant cell containing the encoding gene of the 3-O-sulfate transferase mutant SM1 as described in claim 1.
5. The recombinant cell as described in claim 4, characterized in that, The recombinant cells were obtained by transforming the encoding gene of the 3-O-sulfate transferase mutant SM1 into host cells; the host cells were Escherichia coli Origami B (DE3) containing the molecular chaperone pGro7 helper vector.
6. A method for producing the 3-O-sulfate transferase mutant SM1 by fermentation, characterized in that, The method is as follows: culture the above recombinant cells, and then isolate and obtain the 3-O-sulfate transferase mutant SM1.
7. The method for producing the 3-O-sulfate transferase mutant SM1 by fermentation as described in claim 6, characterized in that, The specific steps are as follows: recombinant cells containing the encoding gene of the 3-O-sulfate transferase mutant SM1 are cultured in LB liquid medium until the OD600 is 0.6-0.8, and IPTG is added to induce expression for 16-18 hours; the cells are collected, sonicated, centrifuged and filtered, and purified by MBP tag to obtain the 3-O-sulfate transferase mutant SM1.
8. The use of the 3-O-sulfatyltransferase mutant SM1 according to claim 1 in the synthesis of 3-O-sulfated heparin oligosaccharides or polysaccharides.
9. The application as described in claim 8, characterized in that, The method uses PAPS as a donor and heparin oligosaccharides and polysaccharides with a GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP structure as the acceptor substrate as the initiating acceptor to catalyze the reaction to generate 3-O-sulfated heparin oligosaccharides or polysaccharides with the structure GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S--GlcA-pNP.