Helicobacter pylori fucosyltransferase mutants

Abstract

The application discloses a mutant of Helicobacter pylori alpha-1,3-fucosyltransferase, a polynucleotide for coding the mutant, an expression vector and a host cell containing the polynucleotide, and further discloses a method for marking a target molecule on a cell or a protein of interest by using the mutant of Helicobacter pylori alpha-1,3-fucosyltransferase, a cell or a protein marked according to the method, and an application of the cell or the protein in preparing a disease treatment drug. The mutant of Helicobacter pylori alpha-1,3-fucosyltransferase provided by the application can mark a target molecule on a cell membrane surface with a better efficiency compared with a wild type, and a method for quantitatively detecting enzyme activity and an application prospect of the marked cell or protein in preparing a disease treatment drug are provided.

Description

Technical Field

[0001] This invention discloses a fucosyltransferase mutant, belonging to the field of enzyme engineering technology. Background Technology

[0002] Glycosylation is a common post-translational modification, a process in which sugars are attached to amino acids or lipids under the control of enzymes. Unlike the biosynthetic pathways of proteins and nucleic acids, glycosylation is a post-translational modification of proteins, a non-template-driven process. Fucosylation is a common type of glycosylation modification in organisms, widely present on the plasma membrane of various cell surfaces, and is considered the endpoint of the glycosylation process. Fucosyltransferases are enzymes that transfer L-fucose from GDP-fucose (guanosine diphosphate fucose) to glycans, participating in the synthesis of terminal glycan structures. Based on the position of fucose at the glycan linker, fucosylation is divided into core fucosylation and branched fucosylation. In Helicobacter pylori, α-1,3-fucosyltransferase catalyzes branched fucosylation, transferring fucose from GDP-fucose (donor substrate) to GlcNAc (N-acetylglucosamine) and LacNac (N-acetyllactosamine) on the N-polysaccharide branch (acceptor substrate). Therefore, fucosyltransferases recognize both donor and acceptor substrates and catalyze the transfer of fucose. According to current literature, the main donor substrate for fucosyltransferases is GDP-fucoose, which has a relatively small molecular weight. Enzyme activity assay kits typically use high-purity GDP-fucoose as the donor substrate, and the acceptor substrate is generally a purified single protein such as fetal globulin (as in assay kits like Promega, catalog numbers VA1090, VA1091, VA1092), with enzyme activity (Km) ranging from approximately 1 to 100 μM. However, when the donor substrate becomes a larger molecular weight GDP-fucoose derivative, or when the acceptor substrate is a complex mixture of proteins (e.g., cell membrane), the enzyme activity (Km) is very low, and the aforementioned commercial assay kits cannot detect this enzyme activity.

[0003] Wu et al. used fucosyltransferases from Helicobacter pylori to transfer antibody-like macromolecular proteins to polysaccharides on the cell membrane surface, such as LacNAc and α2,3 sialylLacNAc. Using this technique, Wu et al. constructed two types of engineered cells—using the natural killer cell line (NK-92MI) and primary mouse CD8+OT-1T cells—and transferred Her2 antibody and PD-L1 antibody to NK-92MI and CD8+OT-1T cells, respectively, via fucosyltransferases. These cells demonstrated specific tumor targeting and inhibitory signals against tumor cells in a mouse model (see Li J, et al. ACS Cent Sci. 2018 Dec 26;4(12):1633-1641.). Therefore, using fucosyltransferases to label therapeutically significant molecules onto cells will significantly enhance the efficacy of cell therapies such as CAR-T. The inventors discovered in their research that, compared to small-molecule GDP-Fucose, fucosyltransferase has nearly a thousand times less enzyme activity for large-molecule donor substrates, which cannot meet the requirements for engineered cell preparation.

[0004] Although several patent documents in this field describe substitution mutations or combinations of substitution mutations that can enhance the activity of α-1,3-fucosyltransferase in Helicobacter pylori, such as CN201611051529.1 and CN202110273518.2, experiments have shown that these mutants, such as mutant A128N15 (A128N, H129E, S46F, Y132I), are not significantly expressed in Escherichia coli BL21(DE3); or they are expressed as inclusion bodies, such as mutants (A78S, T88A, G95V), mutants (F9Y, V19A), and mutants (G224K, V284L). Enzymes expressed in inclusion bodies are difficult to renature, and the activity after renaturation is very low (data not shown). The research and industrial value of these mutants is relatively low. For engineered cell preparation using fucosyltransferase as a tool enzyme, the enzyme needs to be able to better recognize complex substrates.

[0005] Therefore, the purpose of this invention is to obtain a highly active mutant that can be solublely expressed in Escherichia coli and recognize complex substrates, further improving the activity of Helicobacter pylori α-1,3-fucosyltransferase for macromolecular donor substrates and complex acceptor substrates, and to provide corresponding detection methods, thereby expanding the possibility and affordability of preparing engineered cells. Summary of the Invention

[0006] To achieve the above objectives, this invention first provides a mutant of Helicobacter pylori α-1,3-fucosyltransferase, wherein the α-1,3-Helicobacter pylori fucosyltransferase mutant has a mutation at amino acid positions 108 and / or 139 of the wild-type Helicobacter pylori α-1,3-fucosyltransferase sequence shown in SEQ ID NO.1. To obtain the above-mentioned Helicobacter pylori α-1,3-fucosyltransferase, the technical solution adopted by this invention is as follows:

[0007] Using the error-prone PCR mutation principle, a random substitution mutant of α-1,3-fucosyltransferase from *Helicobacter pylori* was obtained based on a truncated version of the α-1,3-fucosyltransferase (strain ATCC700392 / 26695) (sequence shown in SEQ ID NO. 1). SEQ ID NO. 1 is designated as wild-type (wt) in this invention. Both the wild-type and mutant were induced to express in *E. coli*. The soluble mutant was then purified.

[0008] In a preferred embodiment of the Helicobacter pylori α-1,3-fucosyltransferase mutant, the sequence of the mutant is shown in SEQ ID NO.5. Compared to the wild type, the mutant has alanine at position 108 replaced by valine, and in this invention, the mutant is named "A108V".

[0009] The present invention also provides a preferred technical solution for a polynucleotide encoding the above-mentioned Helicobacter pylori α-1,3-Helicobacter pylori fucosyltransferase mutant, the sequence of which is shown in SEQ ID NO.6.

[0010] This invention also provides an expression vector expressing the above-mentioned Helicobacter pylori α-1,3-fucosyltransferase mutant, wherein the expression vector contains the aforementioned polynucleotide. In one specific embodiment of this invention, the vector is a commercially available MilliporeSigma™ pET-41a(+) DNA Vector; other conventional expression vectors in the field of genetic engineering can also be used in this invention.

[0011] This invention also provides a host cell expressing the above-described Helicobacter pylori α-1,3-fucosyltransferase mutant, wherein the host cell contains the above-described expression vector. In one specific embodiment of this invention, the host cell is Escherichia coli BL21(DE3), but other conventional host cells in the field of genetic engineering can also be used in this invention.

[0012] In another preferred embodiment of the Helicobacter pylori α-1,3-fucosyltransferase mutant, the sequence of the mutant is shown in SEQ ID NO.7. Compared to the wild type, the mutant has glutamic acid substituted for aspartic acid at position 139. In this invention, the mutant is named "D139E".

[0013] The present invention also provides a preferred technical solution for a polynucleotide encoding the above-mentioned Helicobacter pylori α-1,3-Helicobacter pylori fucosyltransferase mutant, the sequence of which is shown in SEQ ID NO.8.

[0014] This invention also provides an expression vector expressing the above-mentioned Helicobacter pylori α-1,3-fucosyltransferase mutant, wherein the expression vector contains the aforementioned polynucleotide. In one specific embodiment of this invention, the vector is a commercially available MilliporeSigma™ pET-41a(+) DNA Vector; other conventional expression vectors in the field of genetic engineering can also be used in this invention.

[0015] This invention also provides a host cell expressing the above-described Helicobacter pylori α-1,3-fucosyltransferase mutant, wherein the host cell contains the above-described expression vector. In one specific embodiment of this invention, the host cell is Escherichia coli BL21(DE3), but other conventional host cells in the field of genetic engineering can also be used in this invention.

[0016] Secondly, this invention provides a method for labeling target molecules on target cells or target proteins using the above-mentioned mutant of Helicobacter pylori α-1,3-fucosyltransferase, the method comprising:

[0017] (1) GDP-fucose was conjugated with the target molecule to obtain a linker complex;

[0018] (2) In the presence of the mutant of Helicobacter pylori α-1,3-fucosyltransferase, the ligation complex obtained in step (1) is incubated with the target cell or target protein containing the GlcNac receptor molecule to obtain the target cell labeled with the target molecule.

[0019] In a preferred embodiment, the target molecule is a therapeutic molecule. In one specific embodiment of the invention, the therapeutic molecule is a human IL-15 Fc fusion protein derivative (referred to as N803 in this invention). In this specific embodiment, GDP-fucose is coupled to purified N803 via crosslinking of N-hydroxysuccinimide ester (NHS ester) with an amine. The resulting N803-GDP-fucose conjugate (with a molecular weight of approximately 120 KD; compared to GDP-Fucose with a molecular weight of 500 KD, this is referred to as a high molecular weight GDP-Fucose derivative) serves as the donor substrate. The conjugated N803 carries approximately 6 to 10 GDP-fucose derivatives. In this specific embodiment, the target cell containing the GlcNac receptor molecule is a human washed erythrocyte. Using human washed erythrocytes as the receptor substrate, and catalyzed by a mutant of Helicobacter pylori α-1,3-fucosyltransferase provided in this invention, the GDP-fucose-conjugated N803 is labeled on the erythrocyte membrane surface.

[0020] Third, this invention provides cells or proteins labeled according to the above method. In one specific embodiment of this invention, erythrocytes labeled with GDP-fucose-conjugated N803 on their cell membrane surface were obtained. N803 is an IL-15 derivative that can activate T lymphocytes, B lymphocytes, and NK cells, and mediate the proliferation and survival of these cells, playing an important role in anti-tumor, pro-inflammatory, and anti-infective processes. N803 was developed by Altor BioScience, and a Phase 1 clinical trial showed that Nivolumab (PD-1 antibody, Opdivo) combined with N803 significantly prolonged the long-term survival of patients with metastatic non-small cell lung cancer. As a recombinant protein drug, N803 extends the serum half-life of IL-15 from several minutes to 8-10 hours by fusing with the Fc fragment of an immunoglobulin. In vivo studies in mice have shown a significant improvement in tumor treatment efficacy (Xu H, et al. A novel multimeric IL15 / IL15Rα-Fc complex to enhance cancer immunotherapy. Oncoimmunology. 2021 Mar 11;10:1893500.). However, compared to other tumor immunotherapy drugs, the half-life still needs further improvement. The lifespan of mature human red blood cells is approximately 120 days. Coupling N803 to the surface of red blood cell membranes holds promise for significantly increasing the drug's half-life and reducing the frequency and dosage of intravenous infusions.

[0021] Finally, this invention provides the application of the aforementioned cells or proteins in the preparation of therapeutic drugs. By conjugating drugs to the surface of mature erythrocyte membranes, the half-life, distribution, metabolism, and excretion characteristics of the drugs are altered, thereby maximizing the therapeutic properties of the drugs and reducing side effects. Furthermore, the method provided by this invention can conjugate various therapeutic drugs, including peptides, small molecules, and nucleic acids, to the cell membranes of NK cells, T cells, B cells, as well as stem cells and progenitor cells. This not only preserves the functions of each cell type but also leverages the characteristics of the conjugated drugs to achieve targeted therapy.

[0022] In a preferred embodiment, the disease is a tumor, an inflammatory disease, a metabolic disease, or a rare disease requiring enzyme replacement therapy. For example, antibodies against tumor-associated antigens (TAAs), such as anti-Her2, EFGR, VEGFR, and CD19, can be conjugated to the surface of NK cells to enhance NK cell targeting and improve anti-tumor efficacy. Furthermore, conjugation with one or more tumor-specific recognition antigens can improve the targeting of CAR-T therapy and reduce off-target side effects. Additionally, metabolically related enzymes, such as uricase, can be conjugated to the surface of blood cells to remove uric acid from the blood and tissues, treating refractory gout. Conjugation with immunosuppressive antibodies or peptides, such as PD-L1, can inhibit pathologically activated T lymphocytes and B lymphocytes, thereby treating autoimmune diseases such as lupus; or activating Treg cells to treat inflammatory diseases such as rheumatoid arthritis.

[0023] This invention utilizes the principle of error-prone PCR mutation to screen and obtain random substitution mutants A108V and D139E of α-1,3-fucosyltransferase from wild-type Helicobacter pylori, based on a truncated form of α-1,3-fucosyltransferase. Under the same substrate and experimental conditions, mutants D139E and A108V exhibited significantly higher catalytic efficiency in coupling N803 to the cell membrane surface, with catalytic efficiency 1.75-1.82 times that of the wild type. Other mutants (N167Q and P64S) showed significantly reduced efficiency, only 25%-6% of the wild type. Western blot analysis also confirmed that erythrocytes catalyzed by mutants D139E and A108V carried more N803. Semi-quantitative comparison using grayscale analysis showed that the N803 carried by mutants D139E and A108V was 2 times and 5 times that of the wild type, respectively. The average fluorescence intensity (MFI) of flow cytometry was used as an indicator of the enzyme catalytic reaction product to quantitatively evaluate the enzyme activity of the mutant. The Km of the mutant D139E was about 1 μM, and the enzyme activity was 3 times higher than that of the wild type (about 3 μM). Attached Figure Description

[0024] Figure 1 List of mutations generated by error-prone PCR, amino acids 1-120 (stop codon mutations and frameshift mutations removed).

[0025] Figure 2 List of mutations generated by error-prone PCR, amino acids 121-240 (stop codon mutations and frameshift mutations removed).

[0026] Figure 3 List of mutations generated by error-prone PCR, amino acids 241-364 (stop codon mutations and frameshift mutations removed).

[0027] Figure 4 Detection diagram of soluble expression of mutant in E. coli

[0028] Figure 5 Flow cytometry images of wild-type and mutant-catalyzed macromolecular donor substrate-labeled human erythrocytes;

[0029] Figure 6 Comparison of mean fluorescence intensity of macromolecular donor substrate labeled human erythrocytes catalyzed by wild type and mutant;

[0030] Figure 7 Western blot plot of relative loading of macromolecular donor substrates catalyzed by wild-type and mutant individuals in human erythrocytes;

[0031] Figure 8 A comparison of enzyme activities between wild-type and mutant strains in a simple system (using high-purity GDP-Fucose as the donor substrate and fetoglobulin as the acceptor substrate);

[0032] Figure 9 A comparative graph showing the transfer of donor (GDP-Fucose derivative) to recipient (cell membrane surface of red blood cells) by different concentrations of mutant D139E using flow cytometry under complex donor and recipient substrate conditions.

[0033] Figure 10 A graph showing the relationship between mean fluorescence intensity (MFI) and the concentration of mutant D139E during flow cytometry.

[0034] Figure 11 A graph comparing the enzyme activities of wild-type, mutant D139E, and mutant N167Q by flow cytometry under complex donor and acceptor substrate conditions. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0036] Example 1. Preparation of α-1,3-fucosyltransferase mutant of Helicobacter pylori

[0037] 1. Screening for mutants

[0038] Error-prone PCR involves adjusting reaction conditions during DNA polymerase amplification to randomly introduce mutations into the target gene at a certain frequency, thereby obtaining random mutants of the protein molecule.

[0039] In this invention, the protein sequence of α-1,3-fucosyltransferase of wild-type Helicobacter pylori is shown in SEQ ID NO. 1. Suzhou Junji Biotechnology Co., Ltd. was commissioned to synthesize template DNA and corresponding primers according to the sequence SEQ ID NO. 2.

[0040] Forward: 5'-CATATGTTTCAGCCGCTGCTG-3'

[0041] Reverse: 5'-GTTTACGCGTAAGTCATCGTAATTG-3'

[0042] The reaction conditions for error-prone PCR are as follows:

[0043] Add 5 μL of 10×TITANIUM Taq Buffer (400 mM Tricine-KOH (pH 8.0 at 25°C), 160 mM KCl, 35 mM MgCl2, 37.5 µg / ml BSA) to a 50 μL PCR system, 1 μL of dGTP (2 mM), 1 μL of 50×Diversify dNTP Mix (10 mM each of dATP, dCTP, dGTP, and dTTP), 10 µM each of the two primers, 50 ng of DNA template, and 20 units of TITANIUM Taq. Then, bring the final volume to 50 μL with PCR-grade purified water. All reagents mentioned above are from Clontech, catalog number 639141.

[0044] After thoroughly mixing and centrifuging the reaction components, place the PCR tubes in a PCR thermal cycler. PCR reaction parameters are set as follows: 94°C for 30 seconds; 25 cycles (94°C for 30 seconds, 68°C for 1 minute); 68°C for 1 minute; immersion at 4°C.

[0045] The PCR products obtained above were subjected to agarose gel electrophoresis, gel extraction, and purification. Then, according to a 10 μL reaction system: 1 μL (50 ng) of T vector (T vector was pMD™19-T Vector Cloning Kit, Takara Cat. No. 6013) was added, along with an equimolar amount of PCR product. 1 μL of 10×Buffer containing ATP and an appropriate amount of T4 DNA ligase were added, and the volume was brought to 10 μL with ddH2O. Ligation was performed overnight at 16°C. The ligation product was then transformed into *E. coli*, as briefly described below:

[0046] (1) Preheat the LB plate containing Amp, X-Gal and IPTG to 37°C.

[0047] (2) Add 10 μL of ligation product to 100 μL of competent cells and incubate on ice for 30 min.

[0048] (3) Transfer the centrifuge tube to a 42°C water bath and heat shock for 90 seconds. Then, without shaking the centrifuge tube, quickly place it on ice for 2 minutes.

[0049] (4) Add 300 μL of SOC culture medium to a centrifuge tube, mix well with a pipette tip, and gently shake at 37°C and 150 rpm for 60 min.

[0050] (5) Spread 200 μL of transformed bacterial culture evenly onto LB agar plates containing 50 mg / ml Amp, 20 mg / ml X-gal, and 200 mg / ml IPTG. Incubate overnight at 37°C with the plates inverted. Select white colonies and send them to Suzhou Junji Biotechnology Co., Ltd. for sequencing. Based on the sequencing results, remove nonsense mutations, frameshift mutations, and stop codon mutations, and select single mutation sites. Then, synthesize DNA from each site and clone it into pET41a (MilliporeSigma™ pET-41a(+) DNA Vector, catalog number: 70-556-3). The list of mutants is as follows: Figure 1-3 . Figure 1-3 The amino acid sequences of Helicobacter pylori α-1,3-fucosyltransferase are arranged horizontally, one by one; the vertical axis represents the 20 essential amino acids for humans. The x-axis of the intersection points corresponds to the wild-type amino acid at that site, and the y-axis represents the mutations caused by error-prone PCR.

[0051] 2. Expression of mutants

[0052] The expression of the α-1,3-fucosyltransferase mutant of Helicobacter pylori is briefly described below.

[0053] (1) Take 2 μL of plasmid and add it to 100 μL of BL21(DE3) competent cells (ThermoFisher, catalog number: EC0114), mix immediately, and place on ice for 30 min.

[0054] (2) Heat shock at 42℃ for 90 s, followed by rapid ice bath for 2 min.

[0055] (3) Add 500 uL LB medium and incubate at 37℃ with shaking at rpm<=200 for 60 min.

[0056] (4) Centrifuge at 6000 rpm for 1 min, discard most of the supernatant, leaving about 100-150 uL. Resuspend the bacterial cells and spread them on LB plates containing Amp. Incubate overnight at 37°C.

[0057] (5) Small-scale expression: Pick one single clone into 1 mL of Amp-resistant LB medium and culture at 37 ℃ and 220 rpm for about 5 h. Add 2.5 mL of LB liquid medium containing Amp to the tube from the previous step and culture at 37 ℃ and 220 rpm overnight.

[0058] (6) Transfer the overnight cultured bacterial solution to 20 ml of LB medium containing Amp at a ratio of 1:50, and incubate at 37°C and 220 rpm until OD600 = 0.6 (about 3 h). Add 0.5 mM IPTG to the final concentration and incubate at 30°C and 220 rpm for 6 h.

[0059] (7) Measure the OD600 of the culture medium. Take 10OD of bacterial culture, centrifuge at 10000rpm for 2min, and remove the supernatant.

[0060] (8) Resuspend the bacterial cells in 1 mL of lysis buffer (10 mM Tris-HCl, pH 8.0) and place them on ice for sonication lysis. Sonication conditions: 130 W, 4 min, on 3 s, off 3 s.

[0061] (9) After sonication, the lysis solution was centrifuged at 12000 rpm for 10 min to obtain the supernatant; the supernatant was then ultracentrifuged at 125000 g at 4°C.

[0062] Figure 4The image shows the SDS-PAGE spectra of the soluble fractions of all mutants after induction with *E. coli*. 80 μL of purified recombinant protein was added to 20 μL of 5× Reduce loading buffer and heated at 95 °C for 5 min. 12.5 μL (equivalent to 0.1 OD) of each sample was then subjected to SDS-PAGE electrophoresis. The insoluble fractions were also subjected to SDS-PAGE electrophoresis after adding 50 μL of 1× Reduce loading buffer and heating at 95 °C for 5 min (results not shown). The SDS-PAGE results showed that only the P64S, A108V, D139E, and N167Q mutants exhibited visible soluble expression. Figure 4 Samples 46, 47, 53, and 60 were identified as four mutants: P64S, A108V, D139E, and N167Q, respectively. Specifically, P64S refers to the substitution of proline at position 64 with serine; A108V refers to the substitution of alanine at position 108 with valine; D139E refers to the substitution of aspartic acid at position 139 with glutamic acid; and N167Q refers to the substitution of asparagine at position 167 with glutamine.

[0063] 3. Enzyme and protein purification

[0064] Following the expression induction conditions described above, the culture volume was increased to 100 ml, and the supernatant of the sonicated lysis buffer was obtained. According to the user manual, the supernatant was applied to a HiTrap chelate HP column and eluted with 20 mM imidazole solution. The eluent was collected and the solvent was replaced with 50 mM Tris buffer (pH 8.0) by dialysis. Further purification was then performed using gel filtration chromatography (Superdex 200, GE Healthcare) to obtain a highly pure protein with over 98% homogeneity. Protein concentration was determined using a Bio-Rad protein assay kit based on the Bradford method, using bovine serum albumin as a standard.

[0065] Example 2. Preparation of macromolecular donor substrates

[0066] N803 (also known as ALT803, is a human IL-15 Fc fusion protein derivative) [2013 The IL 15 based superagonist ALT 803 promotes the antigen-independent conversion of memory CD8 T cells into innate like effector cells with antitumor]. Based on the published sequence, the corresponding DNA was designed and fused into the human IgG4-Fc sequence. Suzhou Junji Biotechnology Co., Ltd. was commissioned to synthesize plasmid DNA and clone it into the vector pRM293 (pRM293 was obtained by modifying plasmid pTT5, see Shi C. Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293EBNA1 cells. Biochemistry. 2005;44(48):15705–15714.). Suzhou Junji Biotechnology Co., Ltd. was commissioned to transiently transfect the plasmid into HEK293 cells (National Research Council, Canada), and affinity purification was performed using Mabselect sure (Protein A, GE Healthcare). GDP-fucose was coupled to the purified N803 cells via crosslinking of N-hydroxysuccinimide ester (NHS ester) with an amine for later use. Unless otherwise stated, all N803 cells thereafter are GDP-fucose-coupled N803 cells.

[0067] 1. The NHS ester-activated crosslinking agent and the labeled compound react with the primary amine on N803 under physiological to weakly alkaline conditions (pH 7.2 to 9) to form a stable amide bond. Using this principle, the NHS on TCO-PEG4-NHS reacts with the primary amine on the N803 protein to obtain TCO-PEG4-N803. The specific method and process are as follows:

[0068] 400 μg of purified N803 protein was reacted with 150 μg of TCO-PEG4-NHS (Shanghai Pairui Pharmaceutical Technology Co., Ltd., product number A34125), and 20 mM HEPES buffer (pH 7.0~7.5, Thermo Fisher Scientific (China) Co., Ltd., product number 15630) was added. After incubation at room temperature for 30 minutes, 5 μmol Tris buffer was added to terminate the reaction, and the mixture was incubated at room temperature for 5 minutes. The reaction product was then added to a PD SpinTrap G-25 desalting column (Cytiva, product number 28918004), centrifuged at 800×g to remove unreacted and small molecules generated during the reaction, and pure TCO-PEG4-NHS was obtained.

[0069] 2. Based on the inverse Diels-Alder cycloaddition reaction between trans-cyclooctene and tetrazine, a dihydropyridazine bond is formed between TCO (trans-cyclooctene) and Tz (Tetrazine). Utilizing this principle, TCO-PEG4-N803 reacts with the GDP-fucose derivative GDP-Fucose-Triazole-PEG4-Tz to obtain GDP-Fucose-Triazole-PEG4-PEG4-N803, i.e., GDP-fucose-coupled N803. Details are as follows:

[0070] The TCO-PEG4-N803 obtained in the previous step was further reacted with 30 μg GDP-Fucose-Triazole-PEG4-Tz (synthesized by Yantang Biotechnology Co., Ltd., product number YT-HJP-3-29). After incubation at room temperature for 30 minutes, the reaction product was added to a PD SpinTrap G-25 desalting column (Cytiva, product number 28918004), centrifuged at 800×g to remove unreacted and small molecules generated in the reaction, and pure GDP-fucose-coupled N803 was obtained.

[0071] According to MALDI-TOF analysis, the coupled N803 carries approximately 6 to 10 GDP-fucose derivatives. Unless otherwise stated, all subsequent N803s are GDP-fucose coupled N803s.

[0072] Example 3. Mutant-catalyzed macromolecular donor substrate labeling of human erythrocytes and flow cytometry detection

[0073] Obtain 2-3 mL of blood from a healthy, anonymous blood donor via the antecubital vein. After skin disinfection, use a disposable vacuum blood collection device operated by a professional. Allow the blood to flow slowly along the test tube wall, remove the tube at the marked point, and quickly invert it several times to mix and prevent coagulation. Centrifuge the blood at 500×g at 4°C for 5 minutes, and mark the hematocrit (red, lower layer) and plasma (yellow, upper layer) levels on the tube. Slowly and thoroughly aspirate the plasma and buffy coat using a micropipette. Add bleach to the aspirated liquid and discard it into the biohazardous waste disposal. Transfer 0.5 mL of the hematocrit to a 15 mL centrifuge tube, add 5 mL of PBS pH 7.4 solution, cap, and invert several times to mix. Centrifuge at 500×g at 4°C for 5 minutes, aspirate the supernatant, and discard it. Repeat the PBS washing step 3-4 times. Add 1000 μL of PBS to the washed red blood cells and store at 4°C for later use.

[0074] In 5×10 9 100 μg / mL of a fucosyltransferase mutant and 150 μg / mL of GDP-fucose-conjugated N803 were added to erythrocytes and incubated at room temperature for 30 minutes. The cells were washed with PBS pH 7.4, centrifuged at 500×g, and the supernatant was removed. The erythrocytes were resuspended in PBS pH 7.4 and co-incubated with anti-hIgG-Biotin antibody (Beijing Bio-Legend Technology Co., Ltd., product number F030822, 1:200 dilution) at 4°C for 30 minutes. After one wash with PBS, the cells were stained with Streptavidin-PE (BioLegend, Inc., product number 405204, 1:200 dilution) and co-incubated at 4°C for 30 minutes. After one wash with PBS, the cells were detected by flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., product model MateCyte) and analyzed using NovoExpress software.

[0075] like Figure 5 As shown, under the same substrate and experimental conditions, mutants D139E, A108V, N167Q, P64S, and the wild type were all able to couple GDP-fucose-conjugated N803 to the erythrocyte membrane surface, but with varying efficiencies. Mutants D139E and A108V were able to couple more N803 to the cell membrane surface, with efficiencies 1.75-1.82 times that of the wild type, while mutants N167Q and P64S showed significantly reduced efficiencies, only reaching 25%-6% of the wild type. Figure 6 ).

[0076] Example 4. Western blot method for detecting the presence and relative load of antibodies on erythrocyte membranes.

[0077] To further confirm that N803 was transferred to the erythrocyte membrane, the presence and relative load of antibodies on the erythrocyte membrane were detected using Western blotting. Erythrocytes lack a nucleus and organelles and can be lysed in hypotonic solutions. After removing soluble intracellular hemoglobin, the remaining components are membrane proteins. Under the above conditions, 5 × 10⁻⁶ antibodies were catalyzed by mutants of different fucosyltransferases. 7 After hypotonic lysis of red blood cells with purified water, membrane proteins were extracted using a cell membrane protein extraction kit (Beijing Xinquan Yongshuo Technology Co., Ltd., catalog number XQ-P1203). The extraction was performed according to the kit instructions, and is briefly described below: For every 1×10⁻⁶ cells... 7 Add 500 μL of MER reagent to each 100 μL cell pellet, vortex to resuspend, and incubate on ice for 2 min. Transfer the cell suspension to an ice-cold glass homogenizer and manually homogenize 20-30 times in an ice-water bath. Centrifuge the lysate at 800 g, 4°C, for 5 min; the supernatant obtained is the cell membrane-cytoplasm mixture. Add 1 / 10 volume of MER to the supernatant, mix well, incubate on ice for 5 min, and centrifuge at 14000 rpm, 4°C, for 30 min; the precipitate obtained is the cell membrane fraction. Resuspend in 50 μL of resuspending buffer for later use. Add the same volume of 2×SDS-PAGE electrophoresis buffer, take 20 μL of the protein sample separated by PAGE, and transfer it to a nitrocellulose membrane. The cells were incubated with anti-hIgG-Biotin-labeled primary antibody (Bio-Levibio, catalog number F030822) and Streptavidin-HRP (GenScript, catalog number M00091), washed with TBST, and then subjected to chemiluminescence reaction. Images were taken using a ChemiDoc™ imaging system (BioRAD). Untreated red blood cells were used as negative controls and labeled as Naïve cells. Figure 7 ). Figure 7 The results showed that no N803 was detected on untreated red blood cells, nor on red blood cells catalyzed by mutants N167Q and P64S. However, N803 was detected on red blood cells catalyzed by mutants D139E and A108V, as well as on red blood cells catalyzed by the wild-type enzyme, with mutants D139E and A108V carrying significantly more N803. Gray-scale analysis using the ChemiDoc Imagers image analysis software provided with the imaging system showed that mutants D139E and A108V carried 2 times and 5 times more N803 than the wild-type, respectively.

[0078] Example 5. Enzyme activity detection using small molecules and simple proteins as substrates

[0079] During the study, the Promega kit was used to detect the enzyme activity of the four mutants, using high-purity GDP-Fucose as the donor substrate and fetoglobulin as the acceptor substrate. Enzyme activity assays were performed according to the manual (Promega GDP-Glo™ Glycosyltransferase Assay). 100 µM ultrapure GDP-fucose (GDP-Fucose) (Promega Cat.#VA1097) was used as the donor substrate, and 40 µM fetoglobulin (Promega Cat#V4961) was used as the acceptor substrate. Serial dilutions of purified fucose-transferase mutants (0 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng) were added sequentially. All enzyme reactions were performed in 25 µL white 96-well plates. Incubate at room temperature for 60 minutes, then place in a GloMax® 96 microplate luminescence detector (Cat# E6501) and read the values ​​at room temperature. Plot the data with the enzyme mass in the reaction system on the x-axis and the data read from the luminescence detector on the y-axis. Figure 8 Except for mutant P64S, mutants D139E, A108V3, and N167Q did not show significant differences in enzyme activity compared to the wild type.

[0080] Therefore, enzyme activity assays using small molecules (GDP-Fucose) and simple proteins (fetoglobulin) as substrates cannot truly reflect the catalytic capacity of enzymes under complex conditions.

[0081] Example 6. Quantitative analysis of enzyme activity in complex systems using mean fluorescence intensity (MFI) from flow cytometry as an indicator of enzyme reaction products.

[0082] Mean fluorescence intensity (MFI) of flow cytometry can compare the amount of target molecules on the cell membrane under the same conditions and can serve as an indicator of enzyme reaction products. In this invention, fucotransferase is used as a tool enzyme to transfer clinically significant proteins onto the cell membrane. When preparing engineered cells, the substrate of fucotransferase is no longer a single, simple substrate as in ideal conditions, but a complex and non-homogeneous mixture of substrates. For example, the donor substrate used in this invention is GDP-fucose-conjugated N803, and the acceptor substrate is erythrocytes. The detection established in Example 3 compares the amount of labeled molecules on cells under the same conditions (donor concentration, number of acceptors) with wild-type and mutant at the same "single" concentration. It does not fully reflect enzyme activity, but is only a simple comparison. Km cannot be calculated, and it is a qualitative analysis. The purpose is to distinguish and remove inactive mutants. Example 3 ( Figure 5 ) and Example 5 ( Figure 8 The results showed that enzyme mutants exhibited drastically different characteristics in simple and complex systems. For example, the mutant N167Q showed good enzyme activity in simple systems but no activity in complex systems. Therefore, when describing the activity characteristics of tool enzymes required for engineered cell preparation, it is necessary to establish a quantitative enzyme activity analysis method based on complex systems. Therefore, we established a flow cytometry-based quantitative enzyme activity detection method and performed nonlinear regression analysis based on the Michaelis-Menten equation to calculate Km using the fitted equation. In this method, a suitable enzyme concentration is first identified; in this invention, this means "not selecting saturation concentrations and insensitive regions," and data are measured for different concentrations of the substrate. Then, at this enzyme concentration, data on the enzyme reaction products (using MFI as the reaction product data) are measured for different concentrations of substrate (in this invention, the donor substrate GDP-fucose-coupled N803) using the Michaelis-Menten equation. Then, nonlinear regression analysis is performed based on the Michaelis-Menten equation, and Km is calculated using the fitted equation. Km is only related to the properties of the enzyme and is independent of the enzyme concentration.

[0083] First, determine the appropriate enzyme concentration. With substrate in excess, gradually increase the enzyme concentration, then test the efficiency of substrate transfer to cells, and select the enzyme concentration where the efficiency is moderate. The specific process is as follows:

[0084] In 5×10 9 50 μg / mL GDP-fucose-conjugated N803 was added to 1 mL of red blood cells, followed by different concentrations of the D139E mutant of fucosyltransferase: 0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL. The cells were incubated at room temperature for 30 minutes. The cells were washed with PBS (pH 7.4), centrifuged at 500 × g, and the supernatant was removed. Red blood cells were resuspended in PBS (pH 7.4) and incubated with anti-hIgG-Biotin antibody (Beijing Bio-Lab Technology Co., Ltd., product number F030822, 1:200 dilution) at 4°C for 30 minutes. After washing once with PBS, the cells were stained with Streptavidin-PE (BioLegend, Inc., product number 405204, 1:200 dilution) and incubated at 4°C for 30 minutes. After washing once with PBS, the cells were detected by flow cytometry (Beijing Cenglang Biotechnology Co., Ltd., product model MateCyte) and analyzed using NovoExpress software. The mean fluorescence intensity (MFI) of the flow cytometry cells was obtained.

[0085] like Figure 9 and Figure 10As shown, the reaction reached saturation at a concentration of 20 μg / mL for the D139E mutant; therefore, a concentration of 10 μg / mL (80 nM) was selected for Km determination. The procedure is as follows:

[0086] Wild-type, mutant N167Q and mutant D139E concentrations were fixed at 10 μg / mL (80 nM) at 5 × 10⁻⁶ m³ / mL. 9 0 nM (0 μg / mL), ~40 nM (5 μg / mL), ~80 nM (10 μg / mL), ~160 nM (20 μg / mL), 320 nM (40 μg / mL), 640 nM (80 μg / mL), and 1280 nM (160 μg / mL) GDP-fucose-conjugated N803 were added to / mL of red blood cells. Incubation and flow cytometry were performed as described in Example 3. The average fluorescence intensity was obtained, and nonlinear fitting was performed using GraphPadPrism 8 software. The Km value was calculated using the enzyme kinetic analysis and Michaelis-Menten equation provided in the software. Figure 11 This graph shows a comparison of enzyme activities measured by flow cytometry in complex donor and acceptor substrate conditions for wild-type, mutant D139E, and mutant N167Q. Mutant N167Q showed almost no enzyme activity, mutant D139E had a Km of approximately 1 μM, and the wild-type had a Km of approximately 3 μM. The enzyme activity was 3-fold higher than the wild-type Km (approximately 3 μM).

Claims

1. A mutant of Helicobacter pylori α-1,3-fucosyltransferase, characterized in that, The Helicobacter pylori α-1,3-fucosyltransferase mutant is a mutant with an amino acid variation at position A108V or D139E in the wild-type Helicobacter pylori α-1,3-fucosyltransferase sequence as shown in SEQ ID NO.

1.

2. The Helicobacter pylori α-1,3-fucosyltransferase mutant according to claim 1, characterized in that, The sequence of the fucosyltransferase mutant of Helicobacter pylori α-1,3- is shown in SEQ ID NO.

5.

3. A polynucleotide encoding the Helicobacter pylori α-1,3-fucosyltransferase mutant of claim 2, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.

6.

4. An expression vector for expressing the Helicobacter pylori α-1,3-fucosyltransferase mutant of claim 2, characterized in that, The expression vector contains the polynucleotide as described in claim 3.

5. A host cell expressing the Helicobacter pylori α-1,3-fucosyltransferase mutant of claim 2, characterized in that, The host cell contains the expression vector as described in claim 4.

6. The Helicobacter pylori α-1,3-fucosyltransferase mutant according to claim 1, characterized in that, The sequence of the fucosyltransferase mutant of Helicobacter pylori α-1,3- is shown in SEQ ID NO.

7.

7. A polynucleotide encoding the Helicobacter pylori α-1,3-fucosyltransferase mutant of claim 6, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.

8.

8. An expression vector for expressing the Helicobacter pylori α-1,3-fucosyltransferase mutant of claim 6, characterized in that, The expression vector contains the polynucleotide of claim 7.

9. A host cell expressing the Helicobacter pylori α-1,3-fucosyltransferase mutant of claim 6, characterized in that, The host cell contains the expression vector as described in claim 8.

10. A method for labeling target molecules or target proteins on target cells using a mutant of Helicobacter pylori α-1,3-fucosyltransferase as described in claim 2 or 6, characterized in that, The method includes: (1) GDP-fucose was conjugated with the target molecule to obtain a linker complex; (2) In the presence of the mutant of Helicobacter pylori α-1,3-fucosyltransferase, the ligation complex obtained in step (1) is incubated with the target cell or target protein containing the GlcNac receptor molecule to obtain the target cell labeled with the target molecule.

11. The method according to claim 10, characterized in that, The target molecule is a therapeutic molecule.

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