Heparin 6-O-sulfuryl transferase, high-robustness mutant thereof, mutant screening method and application of heparin 6-O-sulfuryl transferase

Abstract

The invention relates to heparin 6-O-sulfate transferase, a high-robustness mutant of heparin 6-O-sulfate transferase, a mutant screening method and an application of heparin 6-O-sulfate transferase. The amino acid sequence of the heparin 6-O-sulfate transferase is as shown in SEQ ID NO.2, and the amino acid sequence of a truncated body of the heparin 6-O-sulfate transferase is as shown in SEQ ID NO.4. The invention also provides a high-robustness heparin 6-O-sulfuryl transferase mutant M10 (SRC), the amino acid sequence of which is as shown in SEQ ID NO.8, and the mutant M10 (SRC) is characterized in that after 22 amino acids are truncated from the N end of heparin 6-O-sulfuryl transferase Om6OST-1, 43 amino acids are mutated. The heparin 6-O-sulfuryl transferase is brand new heparin 6-O-sulfuryl transferase Om6OST-1 which is derived from qiangqiangfish, and has the activity of transferring sulfate radicals to-GlcNS-of a heparin structure. The heparin 6-O-sulfuric acid group transferase truncated body Om6OST-1-delta N22 has the longest half-life period while the activity is maintained. The 6-O-sulfuryl transferase mutant M10 (SRC) has higher activity of transferring sulfate radicals to-GlcNS-of a heparin structure, and the activity of the 6-O-sulfuryl transferase mutant M10 (SRC) is 25.94 times that of a wild type.

Description

Technical Field

[0001] This invention relates to a heparin 6-O-sulfate transferase and its highly robust mutants, mutant screening methods and applications, belonging to the field of enzyme engineering technology. Background Technology

[0002] Heparin is a glycosaminoglycan with diverse physiological activities. It is a linear sulfated polysaccharide composed of uronic acid and glucosamine linked by 1,4 glycosidic bonds. It participates in many fundamental physiological and pathophysiological functions. Heparin can be divided into anticoagulant heparin and non-anticoagulant heparin, and is widely used in various medical fields such as anticoagulation therapy, tumor immunomodulation, and inflammatory response suppression.

[0003] Currently, heparin is mainly produced by extracting it from animal tissues and organs such as bovine lungs and porcine intestinal mucosa. However, traditional animal-derived heparin production suffers from problems such as poor safety, low yield, high solvent consumption, and severe environmental pollution. Therefore, developing non-animal-derived heparin production methods has become an urgent issue to be addressed.

[0004] In recent years, the chemical enzymatic synthesis of heparin oligosaccharides has emerged as a novel synthetic strategy, gradually becoming an ideal method for heparin synthesis due to its advantages such as strong stereoselectivity, high yield, mild reaction, and uniform and stable product quality. This method utilizes recombinant glycosaminoglycan synthase and non-natural uridine diphosphate monosaccharide donors to mimic the in vivo heparin synthesis process, achieving highly efficient oligosaccharide synthesis. Furthermore, chemical enzymatic synthesis can also be used to construct structure-function libraries of heparin and heparin-like molecules, providing support for new drug development.

[0005] Despite the numerous advantages of chemoenzymatic synthesis of heparin oligosaccharides, its development still faces several challenges. First, the required enzyme elements remain a bottleneck restricting the further development of this strategy. Second, impurities and byproducts that may be introduced during synthesis need to be controlled through optimization of reaction conditions and purification methods. Therefore, future research should focus on the development of enzyme elements, optimization of reaction conditions, and improvement of product purity to promote the application of chemoenzymatic synthesis of heparin oligosaccharides.

[0006] In summary, the chemical enzymatic synthesis of heparin oligosaccharides, as an emerging synthetic strategy, has broad application prospects. Through continuous optimization of the synthesis process and improvement of product quality, it is expected to provide a safe, efficient, and controllable alternative for heparin production, meeting clinical needs. However, for the detection of expression levels of different proteins, there is a lack of a method involving ultra-high throughput, high throughput, and low-throughput detection with high accuracy.

[0007] 6-O-sulfotransferase (6OST) is a key enzyme in the sulfation modification of glycosaminoglycans to form important anticoagulant structural units. It can catalyze the transfer of sulfate groups to the C6-OH position of uronic acid. However, the wild-type 6OST currently in use has the disadvantages of low enzyme activity, poor stability, and low expression levels. Therefore, finding isoenzymes or mutants of this enzyme is helpful for the efficient synthesis of heparin backbone glycan chains and for in-depth research on the reaction mechanism of this enzyme family. In addition, it is also helpful to establish a method to detect the expression levels of different proteins to aid in the discovery of mutants. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a heparin 6-O-sulfate transferase, its highly robust mutants, mutant screening methods, and applications.

[0009] The technical solution of this invention is as follows:

[0010] In a first aspect, the present invention provides a novel heparin 6-O-sulfate transferase Om6OST-1, 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;

[0011] The novel heparin 6-O-sulfate transferase is derived from the bluefin bream.

[0012] In a second aspect, the present invention provides a truncated form of heparin 6-O-sulfate transferase, Om6OST-1-ΔN22, the amino acid sequence of which is shown in SEQ ID NO.4 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3;

[0013] The truncated form of heparin 6-O-sulfate transferase, Om6OST-1-ΔN22, is based on the novel heparin 6-O-sulfate transferase Om6OST-1, with 22 amino acids shortened from the N-terminus.

[0014] A third aspect of the present invention provides a highly robust heparin 6-O-sulfate transferase mutant M10 (SRC), the amino acid sequence of which is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7;

[0015] The highly robust heparin 6-O-sulfate transferase mutant M10 (SRC) is based on heparin 6-O-sulfate transferase Om6OST-1, with 22 amino acids truncated at the N-terminus and 43 amino acids mutated. The specific mutations are: Q24D, E28D, Q50R, V52L, V56Q, K75N, G104E, L106M, S111N, Q113P, S115N, L116R, L149R, S169D, and C17. 4V, Q177D, Q178E, T208Y, K212E, K213E, S215N, Q216R, L217I, F243Y, R255K, M258T, S262K, G267S, D269 E, N272P, E287Q, F302Y, Q310E, F313I, S315N, M317E, Q318E, Q319R, M339S, V340Q, K350R, G352E, S376K.

[0016] Compared to heparin 6-O-sulfate transferase Om6OST-1, the highly robust heparin 6-O-sulfate transferase mutant M10 (SRC) has a 22-amino acid truncated at the N-terminus, with the following mutations: glutamyl at position 24 is mutated to aspartic acid, glutamate at position 28 is mutated to aspartic acid, glutamyl at position 50 is mutated to arginine, valine at position 52 is mutated to leucine, valine at position 56 is mutated to glutamyl, lysine at position 75 is mutated to aspartic acid, glycine at position 104 is mutated to glutamate, and leucine at position 106 is mutated to methylthiocyanate. The following mutations occurred: 111 serine was mutated to asparagine; 113 glutamine was mutated to proline; 115 serine was mutated to asparagine; 116 leucine was mutated to arginine; 149 leucine was mutated to arginine; 169 serine was mutated to aspartic acid; 174 cysteine ​​was mutated to valine; 177 glutamine was mutated to aspartic acid; 178 glutamine was mutated to glutamic acid; 208 threonine was mutated to tyrosine; 212 lysine was mutated to glutamic acid; and 213 lysine was mutated to tyrosine. The following mutations occur: glutamic acid at position 215, serine at position 215 becomes asparagine at position 216, glutamine at position 217 becomes isoleucine at position 217, phenylalanine at position 243 becomes tyrosine at position 255, arginine at position 258 becomes isoleucine at position 258, serine at position 262 becomes lysine at position 267, glycine at position 267 becomes serine at position 269, aspartic acid at position 269 becomes glutamic acid, asparagine at position 272 becomes proline, glutamic acid at position 287 becomes glutamine at position 302... The following mutations were made: phenylalanine was mutated to tyrosine; glutamine at position 310 was mutated to glutamic acid; phenylalanine at position 313 was mutated to isoleucine; serine at position 315 was mutated to asparagine; methionine at position 317 was mutated to glutamic acid; glutamine at position 318 was mutated to glutamic acid; glutamine at position 319 was mutated to arginine; methionine at position 339 was mutated to serine; valine at position 340 was mutated to glutamine; lysine at position 350 was mutated to arginine; glycine at position 352 was mutated to glutamic acid; and serine at position 376 was mutated to lysine.

[0017] A fourth aspect of the present invention provides a recombinant vector constructed by inserting the nucleotide sequences of the heparin 6-O-sulfate transferase Om6OST-1, the truncated heparin 6-O-sulfate transferase Om6OST-1-ΔN22, or the heparin 6-O-sulfate transferase mutant M10 (SRC) into a plasmid vector. The recombinant vector is not particularly limited to a starting 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] More preferably, the plasmid vector is pMAl(c5x).

[0019] A fifth aspect of the present invention provides a recombinant bacterial strain obtained by transforming the aforementioned recombinant vector 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 coding gene of the mutant of the present invention, and is called a recombinant host cell after introduction. The bacterial strain of the present invention can be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, and more preferably Escherichia coli Origami B (DE3).

[0020] A sixth aspect of the present invention provides a method for screening the above-mentioned highly robust heparin 6-O-sulfate transferase mutant M10 (SRC), comprising the following steps:

[0021] The three-dimensional protein structure of the heparin 6-O-sulfate transferase shown in SEQ ID NO.2 was predicted using SWISS-MODEL, and the mutation sites of the obtained protein three-dimensional structure were calculated and predicted using PROSS software to obtain the pseudo-mutant sites.

[0022] A mutant gene containing a proto-mutant site was artificially synthesized, and its activity was measured after heterologous expression to screen for the first generation of mutants. Then, the first generation of mutants were subjected to combinatorial mutagenesis, artificial gene synthesis, heterologous expression, and activity measurement to screen for the second generation of mutants. Rare codons were then randomly introduced into the second generation of mutants to construct a mutant library. Finally, the mutant library was screened by one round of flow cytometry, two rounds of ELISA screening, and one round of enzyme activity measurement to ultimately screen for the mutant with the best enzyme activity and the highest expression level.

[0023] According to a preferred embodiment of the present invention, the activity assay refers to: constructing a reaction system using the heparin backbone pentasaccharide of GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP as the acceptor substrate, PAPS as the donor substrate, and the enzyme to be screened as the catalytic enzyme; then placing the reaction system in a 37°C water bath for 1.5 h, terminating the reaction by heating with boiling water to inactivate the enzyme, and filtering the reaction solution through a 0.22 μm filter membrane before HPLC detection.

[0024] According to a preferred embodiment of the present invention, the flow cytometry screening refers to: ligating the fluorescent reporter gene GFP with the mutant library gene and then introducing them together into the host cell to obtain recombinant cells; then using flow cytometry to analyze the recombinant cells and judging the expression level of the enzyme to be screened based on the intensity of its green fluorescence.

[0025] According to a preferred embodiment of the present invention, the enzyme-linked immunosorbent assay (ELISA) screening refers to: extracting mutants with high expression levels from recombinant cells obtained by flow cytometry screening, detecting them under conditions of excitation wavelength of 488 nm and emission wavelength of 510 nm, and judging the expression level of the enzyme to be screened based on the absorbance data.

[0026] A seventh aspect of the present invention provides the use of the above-mentioned heparin 6-O-sulfate transferase Om6OST-1, the truncated heparin 6-O-sulfate transferase Om6OST-1-ΔN22, or the heparin 6-O-sulfate transferase mutant M10 (SRC) in the synthesis of heparin glycans.

[0027] Experimental procedures not described in detail in this invention can be performed according to conventional experimental procedures in this technical field.

[0028] Beneficial effects

[0029] 1. The heparin 6-O-sulfate transferase Om6OST-1 disclosed in this invention is a novel heparin 6-O-sulfate transferase Om6OST-1 derived from the bluefin bream, possessing the activity of transferring sulfate to the -GlcNS- group of the heparin structure. This expands the sources of heparin 6-O-sulfate transferases and provides a novel heparin 6-O-sulfate transferase for research and use.

[0030] 2. The heparin 6-O-sulfate transferase truncated form Om6OST-1-ΔN22 provided by this invention, compared to wild-type heparin 6-O-sulfate transferase Om6OST-1, involves the deletion of a meaningless fragment at the N-terminus of the wild-type Om6OST-1 protein, resulting in the deletion of 22 amino acids at the N-terminus. This heparin 6-O-sulfate transferase truncated form Om6OST-1-ΔN22 retains its activity in transferring sulfate from the -GlcNS- group of the heparin structure. Compared to wild-type heparin 6-O-sulfate transferase Om6OST-1, the truncated form Om6OST-1-ΔN22 exhibits the longest half-life while maintaining its activity.

[0031] 3. The 6-O-sulfate transferase mutant M10 (SRC) disclosed in this invention is a novel artificial heparin 6-O-sulfate transferase. Compared with the wild-type heparin 6-O-sulfate transferase Om6OST-1, the N-terminus is shortened by 22 amino acids and a 43-amino acid site mutation occurs, resulting in higher activity for transferring sulfate to the -GlcNS- structure of heparin, which is 25.94 times that of the wild-type heparin 6-O-sulfate transferase Om6OST-1. Preliminary stability tests show that the stability of the 6-O-sulfate transferase mutant M10 (SRC) is 7 times higher than that of the wild-type heparin 6-O-sulfate transferase Om6OST-1, making it more suitable for industrial use and large-scale production of heparin. This greatly promotes the development of heparin biomimetic synthesis and application, and opens a new chapter in the research and development of glycosaminoglycans.

[0032] 4. Obtaining more target metabolites from a single enzyme conversion is crucial for screening highly active enzymes. This necessitates detecting both enzyme expression levels and the expression levels of target metabolites. Traditional, well-known enzyme quantification methods include SDS-PAGE electrophoresis and Western blotting, which are time-consuming and labor-intensive. This invention, based on high-performance liquid chromatography (HPLC) enzyme activity detection and microplate reader detection, uses green fluorescent protein emission to indicate enzyme expression levels. This method is both accurate and simple, significantly improving detection efficiency and demonstrating greater application potential. Attached Figure Description

[0033] Figure 1 The results of SDS-PAGE analysis of the purified 6-O-sulfate transferase Om6OST-1 from Escherichia coli are shown.

[0034] In the figure: M is the marker; Om6OST-1 is the recombinant protein of the purified 6-O-sulfate transferase Om6OST-1.

[0035] Figure 2 The liquid phase result is shown for the reaction of 6-O-sulfate group transfer Om6OST with the heparin pentose skeleton.

[0036] Figure 3 The results of the comparison of enzyme activity and stability of different truncated forms of 6-O-sulfotransferase and wild-type 6-O-sulfotransferase Om6OST-1;

[0037] In the figure: the vertical axis represents wild-type 6-O-sulfate transferase Om6OST-1 and different truncated forms of 6-O-sulfate transferase, and the horizontal axis represents enzyme activity and half-life.

[0038] Figure 4 Comparison of enzyme activity and expression levels between the truncated 6-O-sulfotransferase Om6OST-ΔN22 and different 6-O-sulfotransferase mutants;

[0039] In the figure: the horizontal axis represents the truncated 6-O-sulfate transferase Om6OST-ΔN22 and different 6-O-sulfate transferase mutants, and the vertical axis represents enzyme activity and expression level.

[0040] Figure 5 The enzyme activity comparison results of 14 mutants obtained by iterative combination complementation mutation of 6-O-sulfotransferase mutant M4;

[0041] In the figure: the horizontal axis represents different mutants, and the vertical axis represents enzyme activity.

[0042] Figure 6 Comparison of enzyme activity and stability of the truncated 6-O-sulfotransferase Om6OST-ΔN22, the 6-O-sulfotransferase mutants M9, M9-D103P / S267G, and M9-D103P / E273A;

[0043] In the figure: the horizontal axis represents the mutant, and the vertical axis represents enzyme activity and half-life.

[0044] Figure 7 Flow cytometry screening results for recombinant cells carrying the GFP reporter gene;

[0045] In the figure: the horizontal axis represents fluorescence intensity, and the vertical axis represents cell count.

[0046] Figure 8 These are the results of the first round of ELISA reader screening;

[0047] In the figure: the horizontal axis represents the mutant, and the vertical axis represents the fluorescence intensity.

[0048] Figure 9 The results of the second round of ELISA reader screening;

[0049] In the figure: the horizontal axis represents the mutant, and the vertical axis represents the fluorescence intensity.

[0050] Figure 10The results of enzyme activity assays were obtained after one round of flow cytometry screening and two rounds of enzyme-linked immunosorbent assay (ELISA) screening.

[0051] In the figure: the horizontal axis represents the mutant, and the vertical axis represents the multiplier of improvement. Detailed Implementation

[0052] 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 technical means used in the present invention are all methods known to those skilled in the art.

[0053] Example 1: Heterologous expression of a novel 6-O-sulfate transferase

[0054] 1. During a bioinformatics database search using the BLASTp algorithm, the inventors discovered that the amino acid sequence of a gene from *Gymnocypris chinensis* shared 78% homology with the previously reported human 6-O-sulfate transferase 6OST. Therefore, they speculated that the protein product expressed by this gene might possess 6-O-sulfate transfer activity along the heparin backbone. The inventors then optimized the 6-O-sulfate transferase codons in *E. coli*, expressed the protein in an *E. coli* expression system, and named the optimized gene Om6OST-1, whose nucleotide sequence is shown in SEQ ID NO.1. The expressed protein product was named Om6OST-1, and its amino acid sequence is shown in SEQ ID NO.2.

[0055] 2. Nanjing Genscript Biotech Co., Ltd. was commissioned to construct a recombinant vector plasmid pMAL-c5x-6OST containing the Om6OST-1 gene based on the nucleotide sequence information shown in SEQ ID NO.1. This recombinant vector plasmid was then transformed into *E. coli* Origami B(DE3) competent cells, plated on LB solid medium containing carboxybenzyl (50 μg / mL), and cultured at 37°C for 12 h. After screening, positive transformants of Om6OST-1 were obtained. Simultaneously, an empty vector plasmid was transformed and cultured under the same conditions as a negative control.

[0056] Single colonies of Om6OST-1 positive transformants were picked and cultured overnight at 37°C and 225 rpm in 20 mL of sterile LB broth (50 μg / mL carbenicillin). The activated bacterial culture was then inoculated into 1 L of LB broth (50 μg / mL carbenicillin) at a 1% inoculation rate and cultured at 37°C and 225 rpm for 3 h until OD (digesterone) was reached. 600The concentration of the culture medium was approximately 0.6-0.8. IPTG was added to a final concentration of 0.2 mM, and the mixture was induced at 22℃ and 225 r / min for 16-18 h. The bacterial cells were then collected. The cells were resuspended in equilibration buffer (1×PBS, pH=8.00) and sonicated on ice (15 s on, 45 s off, amplitude 33%, energy 1500 KJ, 4℃) for 30 min. The lysed cells were centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane, and the protein was purified using an MBP column. After loading the sample, the cells were washed with equilibration buffer, then washed with washing buffer (1×PBS, pH=8.00) to remove impurities, and finally eluted with elution buffer (1×PBS, 20 mM maltose, pH=8.00) to obtain the target protein. The purified 6-O-sulfate transferase Om6OST-1 was stored in 10% glycerol, aliquoted, and stored at -80℃.

[0057] 3. The purified 6-O-sulfate transferase Om6OST-1 was identified by SDS-PAGE, and the results are as follows: Figure 1 As shown.

[0058] Depend on Figure 1 It can be seen that the heterologous expression of 6-O-sulfate transferase Om6OST-1 was successful, and this method can be used to purify and obtain the recombinant protein of 6-O-sulfate transferase Om6OST-1.

[0059] Example 2: Detection of enzyme activity and expression level of a novel 6-O-sulfate transferase

[0060] The heparin backbone pentasaccharide of GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP (final concentration 0.2 mM) was used as the acceptor substrate, PAPS (final concentration 0.6 mM) as the donor substrate, and the 6-O-sulfate transferase Om6OST-1 prepared in Example 1 as the catalytic enzyme to construct the reaction system, as shown in Table 1. The reaction system was placed in a 37°C water bath overnight, and the reaction was terminated by heating with boiling water for 5 min to inactivate the enzyme. The reaction solution was filtered through a 0.22 μm filter membrane and then analyzed by HPLC. The detection method is shown in Table 2. The mobile phase flow rate was 0.5 mL / min. The obtained chromatographic results are shown below. Figure 2 As shown.

[0061] Table 1

[0062]

[0063] Table 2

[0064]

[0065] Depend on Figure 2It is known that using the heparin pentose backbone of GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP as the initiator acceptor, PAPS as the donor, and 6-O-sulfate transferase Om6OST-1 as the catalytic enzyme, a heparin pentose backbone with the structure GlcA-GlcNS6S-GlcA2S-GlcNS6S-GlcA-pNP can be generated. This indicates that 6-O-sulfate transferase Om6OST-1 possesses the activity of transferring sulfate to the -GlcNS- group of the heparin structure and can be used for heparin glycan synthesis.

[0066] Example 3: Design of the truncated form of 6-O-sulfate transferase, detection of enzyme activity and expression level.

[0067] To further improve the enzyme activity of 6-O-sulfate transferase Om6OST-1, mutation screening was conducted. The specific method is as follows: the three-dimensional protein structure of 6-O-sulfate transferase Om6OST-1 shown in SEQ ID NO.2 was predicted using SWISS-MODEL. The structure was analyzed, and five protein truncated variants were designed, namely, those with 22 amino acids removed from the N-terminus, 34 amino acids removed from the N-terminus, amino acids after the 355th amino acid removed from the C-terminus, amino acids after the 338th amino acid removed from the C-terminus, and amino acids after the 327th amino acid removed from the C-terminus.

[0068] Following the method described in Example 1, nucleotide sequences of five truncated 6-O-sulfate transferases were synthesized, recombinant vector plasmids were constructed, and heterologous expression was performed to obtain the truncated 6-O-sulfate transferases Om6OST-1-ΔN22, Om6OST-1-ΔN35, Om6OST-1-ΔC335, Om6OST-1-ΔC338, and Om6OST-1-ΔC327.

[0069] Finally, following the method described in Example 2, the enzyme activity and half-life of wild-type 6-O-sulfate transferase Om6OST-1 and five 6-O-sulfate transferase truncated variants were determined. The results are as follows: Figure 3 As shown.

[0070] Depend on Figure 3 It can be seen that the truncated form of 6-O-sulfate transferase, Om6OST-1-ΔN22, has the longest half-life while maintaining its activity.

[0071] The amino acid sequence of the truncated 6-O-sulfate transferase Om6OST-1-ΔN22 obtained in this embodiment is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0072] Example 4: Design of 6-O-sulfate transferase mutants, detection of enzyme activity and expression levels.

[0073] To further improve the enzyme activity of the truncated 6-O-sulfate transferase Om6OST-1-ΔN22, mutation screening was performed. The specific method is as follows:

[0074] The three-dimensional structure of the truncated 6-O-sulfate transferase Om6OST-1-ΔN22 shown in SEQ ID NO.3 was predicted using SWISS-MODEL. The mutation sites of the obtained protein three-dimensional structure were calculated and predicted using PROSS software, and the pseudo-mutant sites were obtained, as shown in Table 3.

[0075] Table 3

[0076]

[0077] As can be seen from Table 3, this embodiment designed nine 6-O-sulfate transferase mutants, namely 6-O-sulfate transferase mutants M1 to 9.

[0078] Then, Nanjing GenScript Biotech Co., Ltd. was commissioned to construct recombinant vector plasmids containing the genes of 6-O-sulfotransferase mutants M1-9, based on the nucleotide sequence information shown in SEQ ID NO.3 and the mutation site information shown in Table 3. Heterologous expression was then performed according to the method described in Example 1 to obtain 6-O-sulfotransferase mutants M1-9. Finally, the enzyme activity and expression levels of the truncated 6-O-sulfotransferase Om6OST-1-ΔN22 and the 6-O-sulfotransferase mutants M1-9 were measured according to the method in Example 2. The results are as follows: Figure 4 As shown.

[0079] Depend on Figure 4 It can be seen that the 6-O-sulfate transferase mutant M9 has the best enzyme activity and expression level, and at the same time, it was found that the enzyme activity decreased during the evolution of mutants M3 to M4.

[0080] 2. Using the 6-O-sulfate transferase mutant M4 as the parent, iterative combination and complementation mutations were performed on the four key mutation sites 56, 103, 267, and 273, resulting in 14 6-O-sulfate transferase mutants: Q56V, D103P, S267G, E273A, Q56V / D103P, Q56V / S267G, Q56V / E273A, D103P / S267G, D103P / E273A, S267G / E273A, Q56V / D103P / S267G, Q56V / D103P / E273A, Q56V / S267G / E273A, and Q56V / D103P / S267G / E273A.

[0081] Following the method described in Example 1, the above 14 6-O-sulfate transferase mutants were heterologously expressed. Finally, following the method in Example 2, the enzyme activity and expression levels of the 14 6-O-sulfate transferase mutants were measured, with the data of 6-O-sulfate transferase mutants M3-4 used as controls. The measurement results are as follows: Figure 5 As shown.

[0082] Depend on Figure 5 It can be seen that the enzyme activity of the 6-O-sulfate transferase mutants D103P / S267G and D103P / E273A increased significantly.

[0083] 3. Using the 6-O-sulfate transferase mutant M9 as the parent, the above-obtained mutant combinations were mutated to design two mutants, namely the 6-O-sulfate transferase mutants M9-D103P / S267G and M9-D103P / E273A.

[0084] The two 6-O-sulfate transferase mutants were then heterologously expressed according to the method described in Example 1. Finally, the 6-O-sulfate transferase Om6OST-1, the 6-O-sulfate transferase mutant M9, and the 6-O-sulfate transferase mutants D103P / S267G and D103P / E273A were placed at 37°C, and samples were taken at regular intervals. The reaction system according to Table 1 of Example 2 was placed in a 37°C water bath for 1 hour, and the reaction was terminated by heating with boiling water for 5 minutes to inactivate the enzymes. The reaction solution was filtered through a 0.22 μm filter membrane and then analyzed by liquid chromatography according to the method in Table 2 of Example 2. The results are as follows: Figure 6 As shown.

[0085] Depend on Figure 6It can be seen that, compared with the 6-O-sulfate transferase mutant M9, the enzyme activities of the 6-O-sulfate transferase mutants M9-D103P / S267G and M9-D103P / E273A are further increased. Among them, the enzyme activity of the 6-O-sulfate transferase mutant M9-D103P / E273A is the highest, and it is designated as the 6-O-sulfate transferase mutant M10. The stability of the 6-O-sulfate transferase mutant M10 changes less, with a half-life of about 70 hours, making it more suitable for industrial use and large-scale production of heparin.

[0086] The amino acid sequence of the 6-O-sulfate transferase mutant M10 obtained in this embodiment is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5.

[0087] Example 5: Codon modification to design high-expression mutants

[0088] To further increase the expression level of the 6-O-sulfotransferase mutant, further mutation screening was conducted. The specific methods are as follows:

[0089] Rare codons were randomly introduced into the amino acid sequence of the 6-O-sulfotransferase mutant M10 to construct a mutant library. The mutant library was screened by one round of flow cytometry, two rounds of ELISA, and one round of enzyme activity assay. Finally, the 6-O-sulfotransferase mutant with the best enzyme activity and the highest expression level was selected and designated as 6-O-sulfotransferase mutant M10 (SRC).

[0090] The flow cytometry screening process involves: ligating the fluorescent reporter gene GFP with a mutant library gene and introducing them into host cells to obtain recombinant cells; then analyzing these recombinant cells using flow cytometry, and determining the expression level of the screening enzyme based on the intensity of the green fluorescence. Leveraging the high throughput of flow cytometry, the top 3% of positive cells are selected. Specific results are as follows... Figure 7 As shown.

[0091] The two rounds of ELISA screening refer to: extracting high-expression 6-O-sulfate transferase mutants from recombinant cells obtained by flow cytometry screening, detecting them under excitation wavelength of 488 nm and emission wavelength of 510 nm, and judging the expression level of the enzyme to be screened based on the absorbance data to complete the first round of ELISA screening. The results are as follows: Figure 8 As shown.

[0092] Depend on Figures 7-8It can be seen that the first round of ELISA screening identified 21 6-O-sulfate transferase mutants, namely 6OST-GFP-12, 6OST-GFP-13, 6OST-GFP-14, 6OST-GFP-16, 6OST-GFP-23, 6OST-GFP-25, 6OST-GFP-41, 6OST-GFP-42, 6OST-GFP-45, 6OST-GFP-46, 6OST-GFP-50, 6OST-GFP-54, 6OST-GFP-55, 6OST-GFP-56, 6OST-GFP-57, 6OST-GFP-58, 6OST-GFP-62, 6OST-GFP-64, 6OST-GFP-80, 6OST-GFP-109, and 6OST-GFP-131.

[0093] Then, these 21 6-O-sulfate transferase mutants were further detected under conditions of excitation wavelength of 488 nm and emission wavelength of 510 nm. The expression levels of the enzymes to be screened were judged based on the absorbance data, completing the second round of ELISA screening. The results are as follows: Figure 9 As shown.

[0094] Depend on Figure 9 It can be seen that the second round of ELISA screening identified eight 6-O-sulfate transferase mutants, namely 6OST-GFP-13, 6OST-GFP-14, 6OST-GFP-16, 6OST-GFP-45, 6OST-GFP-46, 6OST-GFP-56, 6OST-GFP-57, and 6OST-GFP-131.

[0095] Finally, the fluorescent reporter gene GFP was removed from these eight 6-O-sulfotransferase mutants, resulting in the 6-O-sulfotransferase mutants 6OST-13, 6OST-14, 6OST-16, 6OST-45, 6OST-46, 6OST-56, 6OST-57, and 6OST-131. Following the method in Example 2, the enzyme activity and expression levels of the eight 6-O-sulfotransferase mutants were measured, with the 6-O-sulfotransferase mutant M10 used as a control. The results are as follows: Figure 10 As shown.

[0096] Depend on Figure 10 It is known that 6OST-13 has the best enzyme activity and the highest expression level, and is named 6OST-M10 (SRC), which is recorded as the highly robust heparin 6-O-sulfate transferase mutant M10 (SRC) of this invention.

[0097] The amino acid sequence of the 6-O-sulfate transferase mutant M10 (SRC) obtained in this embodiment is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A heparin 6-O-sulfate transferase, 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 truncated form of heparin 6-O-sulfate transferase, Om6OST-1-ΔN22, characterized in that, The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence encoding the gene is shown in SEQ ID NO.

3.

3. A highly robust heparin 6-O-sulfate transferase mutant M10 (SRC), characterized in that, The amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence encoding the gene is shown in SEQ ID NO.7; The highly robust heparin 6-O-sulfate transferase mutant M10 (SRC) is based on the heparin 6-O-sulfate transferase Om6OST-1, with 22 amino acids truncated at the N-terminus and 43 amino acids mutated, and the rare codon TAA introduced at nine isoleucine sites. The specific amino acid mutations are: Q24D, E28D, Q50R, V52L, V56Q, K75N, G104E, L106M, S111N, Q113P, S115N, L116R, and L149. R, S169D, C174V, Q177D, Q178E, T208Y, K212E, K213E, S215N, Q216R, L217I, F243Y, R255K, M258T, S262K, G267S , D269E, N272P, E287Q, F302Y, Q310E, F313I, S315N, M317E, Q318E, Q319R, M339S, V340Q, K350R, G352E, S376K.

4. A recombinant vector, characterized in that, The recombinant vector is constructed by inserting the nucleotide sequence of the heparin 6-O-sulfate transferase of claim 1, the truncated heparin 6-O-sulfate transferase Om6OST-1-ΔN22 of claim 3, or the heparin 6-O-sulfate transferase mutant M10 (SRC) of claim 3 into a plasmid vector.

5. A recombinant bacterial strain, characterized in that, The recombinant strain is obtained by transforming the recombinant vector of claim 4 into a host cell.

6. A method for screening highly robust heparin 6-O-sulfatyltransferase mutant M10 (SRC), characterized in that, Includes the following steps: The three-dimensional protein structure of the heparin 6-O-sulfate transferase shown in SEQ ID NO.2 was predicted using SWISS-MODEL, and the mutation sites of the obtained protein three-dimensional structure were calculated and predicted using PROSS software to obtain the pseudo-mutant sites. A mutant gene containing a protomutator site was artificially synthesized, and its activity was measured after heterologous expression to screen for the first generation of mutants. Then, the first generation of mutants were subjected to combinatorial mutagenesis, artificial gene synthesis, heterologous expression, and activity measurement to screen for the second generation of mutants. Rare codons were then randomly introduced into 12 isoleucine sites in the second generation of mutants to construct a mutant library. Finally, the mutant library was screened by one round of flow cytometry, two rounds of ELISA, and one round of enzyme activity measurement to ultimately screen for the mutant with the best enzyme activity and the highest expression level.

7. The screening method as described in claim 6, characterized in that, The activity assay refers to the following steps: using the heparin backbone pentasaccharide of GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP as the acceptor substrate, PAPS as the donor substrate, and the enzyme to be screened as the catalytic enzyme to construct a reaction system; then placing the reaction system in a 37°C water bath for 1 hour, heating with boiling water to inactivate the enzyme and terminate the reaction, and filtering the reaction solution through a 0.22 μm filter membrane before HPLC detection.

8. The screening method as described in claim 6, characterized in that, The flow cytometry screening refers to: ligating the fluorescent reporter gene GFP with the mutant library gene and introducing them together into host cells to obtain recombinant cells; then using flow cytometry to analyze the recombinant cells and judging the expression level of the enzyme to be screened based on the intensity of the green fluorescence.

9. The screening method as described in claim 6, characterized in that, The enzyme-linked immunosorbent assay (ELISA) screening refers to: extracting mutants with high expression levels from recombinant cells obtained by flow cytometry screening, detecting them under conditions of excitation wavelength of 488 nm and emission wavelength of 510 nm, and judging the expression level of the enzyme to be screened based on the absorbance data.

10. The use of the heparin 6-O-sulfate transferase of claim 1, the truncated form of heparin 6-O-sulfate transferase Om6OST-1-ΔN22 of claim 3, or the mutant form of heparin 6-O-sulfate transferase M10 (SRC) of claim 3 in the synthesis of heparin glycans.

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