Curable and reusable helicobacter pylori fucosyltransferase mutant

Abstract

Disclosed in the present invention is a Helicobacter pylori α-1,3-fucosyltransferase mutant, a polynucleotide encoding the mutant, and an expression vector and host cell containing the polynucleotide. Further disclosed in the present invention is a method for site-specific conjugation of the Helicobacter pylori α-1,3-fucosyltransferase mutant to magnetic beads. According to the method, fucosyltransferase can be repeatedly utilized and can be quickly and effectively removed from an enzymatic reaction system. Compared with a wild type, the Helicobacter pylori α-1,3-fucosyltransferase mutant provided by the present invention has a higher yield, and the fucosyltransferase can be effectively fixed on a solid surface, such as the surface of a magnetic bead, by means of a more preferred method without compromising the activity. By means of a magnetic separation method, the fucosyltransferase is separated from the enzymatic reaction system and washed for reuse.

Description

Immobilizable and reusable Helicobacter pylori fucosyltransferase mutant Technical Field

[0001] The invention discloses a fucosyltransferase mutant and belongs to the technical field of enzyme engineering. Background Art

[0002] Helicobacter pylori α-1,3-fucosyltransferase is a branched-chain fucosylation enzyme that transfers fucose from GDP-fucose (donor substrate) to GlcNAc (N-acetylglucosamine) and LacNac (N-acetyllactosamine) (acceptor substrates) on the N-polysaccharide side chains. This reaction is a rapid, unidirectional enzymatic reaction with high regio- and stereoselectivity. Compared to chemical methods, it offers milder conditions and higher yields. However, a major obstacle to industrialization is the rapid separation or removal of the fucosyltransferase after the enzymatic reaction. Although affinity purification using chromatographic columns and membranes can remove the fucosyltransferase, enzyme recovery and reuse remain challenging. In industrial production, excess enzyme is often added to ensure high enzymatic reaction efficiency. Reusing the enzyme not only reduces production costs and minimizes waste, but also reduces the environmental impact of waste. Technical issues

[0003] CN114369585A discloses a recombinant truncated form of Helicobacter pylori α-1,3-fucosyltransferase (FucTd), which exhibits excellent stability and is not consumed or destroyed during the reaction process. This means it can be reused. However, finding a sustainable or optimized utilization process has become a pressing challenge in the industrialization process. The present invention aims to provide a reusable mutant of Helicobacter pylori α-1,3-fucosyltransferase without reducing enzyme activity. Technical Solutions

[0004] To achieve this goal, the present invention utilizes PCR mutagenesis to generate mutants of H. pylori α-1,3-fucosyltransferase (Met1-Asn405-HIS×6, consisting of amino acids from position 1 (Met) to position 405 (Asn) with a purification tag consisting of six histidine residues fused to the C-terminus) based on a recombinant truncated FucTd (sequence shown in SEQ ID NO. 1) of H. pylori α-1,3-fucosyltransferase (strain ATCC 700392 / 26695). The truncated FucTd and mutants were induced to express in E. coli, and the soluble mutants were selected and purified. Based on the above mutation strategy, the present invention first provides a mutant of Helicobacter pylori α-1,3-fucosyltransferase, wherein the amino acid sequence of the mutant of Helicobacter pylori α-1,3-fucosyltransferase is shown in SEQ ID NO.3. In the present invention, the mutant having this sequence is relative to the Helicobacter pylori α-1,3-fucosyltransferase (named "FucTd" in the present invention) with an amino acid sequence as shown in SEQ ID NO.1, and mutations from cysteine ​​(Cysteine) to serine (Serine) occur at amino acids 169 and 238 (C169S and C238S). The mutant is named "FucTd-2C" in the present invention; or,

[0005] The amino acid sequence of the mutant of Helicobacter pylori α-1,3-fucosyltransferase is shown in SEQ ID NO. 5. In the present invention, the mutant having this sequence is a mutation in which a cysteine ​​(406C) is inserted at amino acid position 406 of the Helicobacter pylori α-1,3-fucosyltransferase with respect to the amino acid sequence shown in SEQ ID NO. 1. The mutant is named "FucTd_C406" in the present invention; or

[0006] The amino acid sequence of the mutant of Helicobacter pylori α-1,3-fucosyltransferase is shown in SEQ ID NO.7. In the present invention, the mutant having this sequence is a mutation in which a cysteine ​​(412C) is inserted at amino acid position 412 of the Helicobacter pylori α-1,3-fucosyltransferase with respect to the amino acid sequence shown in SEQ ID NO.1. The mutant is named "FucTd_C412" in the present invention; or

[0007] The amino acid sequence of the mutant of Helicobacter pylori α-1,3-fucosyltransferase is shown as SEQ ID NO.9. In the present invention, the mutant having this sequence is relative to the Helicobacter pylori α-1,3-fucosyltransferase whose amino acid sequence is shown as SEQ ID NO.1, and a mutation of cysteine ​​to serine occurs at amino acids 169 and 238 (C169S and C238S), and a mutation of cysteine ​​(406C) insertion occurs at amino acid 406. The mutant is named "FucTd-2C_C406" in the present invention.

[0008] Secondly, the present invention provides a polynucleotide encoding the Helicobacter pylori α-1,3-fucosyltransferase mutant. Based on the common sense of those skilled in the art and following the triplet code rules for protein coding, the same amino acid has different triplet nucleotide codes. Therefore, any Helicobacter pylori α-1,3-fucosyltransferase mutant that can encode an amino acid sequence such as SEQ ID NO. 3, 5, 7, or 9 is within the scope of the polynucleotide encoding the Helicobacter pylori α-1,3-fucosyltransferase mutant defined in the present invention.

[0009] In a preferred technical solution, the sequence of the polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase whose amino acid sequence is shown in SEQ ID NO.3 is shown in SEQ ID NO.4; or

[0010] The sequence of the polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase whose amino acid sequence is shown in SEQ ID NO.5 is shown in SEQ ID NO.6; or

[0011] The sequence of the polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase whose amino acid sequence is shown in SEQ ID NO.7 is shown in SEQ ID NO.8; or

[0012] The sequence of the polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase whose amino acid sequence is shown in SEQ ID NO.9 is shown in SEQ ID NO.10.

[0013] Third, the present invention provides an expression vector for expressing the aforementioned Helicobacter pylori α-1,3-fucosyltransferase mutant, comprising the aforementioned polynucleotide. In one embodiment, the vector is the commercially available MilliporeSigma™ pET-41a(+) DNA Vector. Other conventional expression vectors in the field of genetic engineering may also be used in the present invention.

[0014] Fourth, the present invention provides a host cell expressing the aforementioned Helicobacter pylori α-1,3-fucosyltransferase mutant, comprising the aforementioned expression vector. In one embodiment of the present invention, the host cell is Escherichia coli BL21(DE3). Other conventional host cells in the field of genetic engineering may also be used in the present invention.

[0015] Fifth, the present invention provides the above-mentioned Helicobacter pylori α-1,3-fucosyltransferase mutant coupled to a solid phase carrier.

[0016] In a preferred embodiment, the solid phase carrier is magnetic beads.

[0017] Sixth, the present invention provides a method for preparing the above-mentioned Helicobacter pylori α-1,3-fucosyltransferase mutant coupled to magnetic beads, the method comprising the following steps:

[0018] (1) Direct coupling to the magnetic bead surface via the free sulfhydryl groups in mutant fucosyltransferases, or,

[0019] (2) The free thiol groups are biotinylated at specific sites and then coupled to the surface of magnetic beads using the biotin-streptavidin affinity method.

[0020] Finally, the present invention provides a method for repeatedly using the Helicobacter pylori α-1,3-fucosyltransferase mutant coupled to a solid phase carrier to transfer a donor substrate coupled to a biomacromolecule to an acceptor substrate, the method comprising the following steps:

[0021] After the GDP-fucose (donor substrate) coupled with the biomacromolecule is transferred to the target cell (acceptor substrate), the magnetic beads are washed in a magnetic field environment, the supernatant is removed, and fresh donor substrate and acceptor substrate are added, and the donor substrate coupled with the biomacromolecule is transferred to the acceptor substrate again.

[0022] In a specific embodiment of the present invention, the donor substrate is GDP-fucose, and the acceptor substrate is a target cell. In a more specific embodiment, the target cell is a NK-92 cell. In another specific embodiment of the present invention, the number of repetitions is 5 times.

[0023] In a preferred embodiment, the biomacromolecule is an antibody. In a specific embodiment of the present invention, the antibody is the Herceptin antibody. Beneficial effects

[0024] The present invention, after performing mutation and expression analysis on the amino acid sequence of a recombinant truncated form of Helicobacter pylori α-1,3-fucosyltransferase (Met1-Asn405-HISx6, FucTd), discovered that the fucosyltransferase contains only two cysteines (Cys) at positions 169 and 238, but these two cysteines do not participate in the enzyme's protein structure by forming disulfide bonds. Mutating these cysteines to other amino acids, such as serine (Ser), did not significantly alter the enzyme's expression level, solubility, or enzyme activity in Escherichia coli.

[0025] Furthermore, retaining the two cysteines at positions 169 and 238 of the recombinant truncated fucosyltransferase (FucTd) and adding additional cysteines at the end of the amino acid sequence did not significantly alter its solubility or enzyme activity, nor did it form aggregates. Instead, the overall expression and yield increased by 3-5 times. This indicates that the two cysteines at positions 169 and 238 are not exposed on the protein molecule surface, and the addition of free cysteines has no effect on yield and enzyme activity. Experiments using maleimide (NHS) to selectively site-specifically conjugate biotin to free sulfhydryl groups on proteins under mild conditions (as shown in Example 5) also confirmed that the two cysteines at positions 169 and 238 do not form adducts with NHS, further demonstrating that the two cysteines at positions 169 and 238 are embedded within the protein molecule. Based on these findings, the present invention utilizes site-directed coupling technology of free cysteine ​​to couple α-1,3-fucosyltransferase mutants of Helicobacter pylori (e.g., FucTd_C406, FucTd_C412, FucT-2C_C406) to the surface of magnetic beads. Compared with the free enzyme, the activity of the fucosyltransferase coupled to the magnetic beads is not only unaffected, but also has an improved catalytic activity compared to the free enzyme. After the fucosyltransferase coupled to the magnetic beads completes the catalytic reaction, the fucosyltransferase can be quickly separated from the reaction system by magnetic separation technology, and after appropriate cleaning, it is continued to be used in a new reaction system. After repeated use for 5 times, the reduction in enzyme activity can be maintained within 4% of the original activity.

[0026] Therefore, this invention can realize the reuse of Helicobacter pylori α-1,3-fucosyltransferase, increase the yield of the target product, reduce production costs and have continuity, and is suitable for continuous and automated production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1. Sequence comparison of mutants of Helicobacter pylori α-1,3-fucosyltransferase;

[0028] Figure 2. SDS-PAGE and size exclusion chromatography (SEC) analysis of mutants of Helicobacter pylori α-1,3-fucosyltransferase;

[0029] Figure 3. Schematic diagram of the GDP-Glo™ glycosyltransferase assay.

[0030] Figure 4. Schematic diagram of flow cytometry detection of glycosyltransferase activity;

[0031] Figure 5. GDP-Glo™ Glycosyltransferase Assay comparing the enzymatic activities of magnetic bead-coupled and free fucosyltransferase mutants.

[0032] Figure 6. Comparison of the enzyme activities of magnetic bead-coupled and free fucosyltransferase mutants by flow cytometry.

[0033] Figure 7. Schematic diagram of repeated use of magnetic beads coupled to fucosyltransferase;

[0034] Figure 8. Repeated use of magnetic bead-coupled fucosyltransferase activity assay. Modes for Carrying Out the Invention

[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 the description proceeds. 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 Helicobacter pylori α-1,3-fucosyltransferase mutants

[0037] 1. Preparation of DNA for Mutants

[0038] Point mutations are achieved by introducing base substitution mutations into the target gene during gene amplification using DNA polymerase in PCR. In the present invention, the DNA sequence of the recombinant truncated (FucTd) Helicobacter pylori α-1,3-fucosyltransferase is shown in SEQ ID NO. 2, and the translated amino acid sequence is shown in SEQ ID NO. 1. The mutation strategy for Helicobacter pylori α-1,3-fucosyltransferase in the present invention is as follows:

[0039] FucTd-2C: CS at position 169 and CS at position 238 of SEQ ID NO. 1 are mutated. The amino acid sequence after mutation is shown in SEQ ID NO. 3, and the DNA sequence is shown in SEQ ID NO. 4.

[0040] FucTd_C406: C is added to position 406 of SEQ ID NO. 1. The amino acid sequence after mutation is shown in SEQ ID NO. 5, and the DNA sequence is shown in SEQ ID NO. 6.

[0041] FucTd_C412: C is added to position 412 of SEQ ID NO. 1. The amino acid sequence after mutation is shown in SEQ ID NO. 7, and the DNA sequence is shown in SEQ ID NO. 8.

[0042] FucT-2C_406C: C-S at position 169, C-S at position 238, and C at position 406 of SEQ ID NO. 1. The amino acid sequence after mutation is shown in SEQ ID NO. 9, and the DNA sequence is described in SEQ ID NO. 10.

[0043] The above recombinant truncations (FucTd) and mutants all carry histone expression tags at the carboxyl terminus for purification of recombinant proteins. FucTd has only two cysteines, all of which were replaced with serines in the present invention to obtain FucT-2C. Secondly, based on FucTd, free cysteines were added through mutations. That is, a cysteine ​​was inserted at the N-terminus (position 406) or C-terminus (position 412) of the histone tag, respectively, to obtain FucT_406C and FucT_412C. Based on FucT-2C, a cysteine ​​was inserted at the N-terminus (position 406) of the histone tag to obtain FucT-2C_406C.

[0044] A schematic diagram of the sequence comparison of the α-1,3-fucosyltransferases of the above four Helicobacter pylori species is shown in FIG1 .

[0045] The DNA shown in SEQ ID NO. 2 was synthesized and cloned into pET41a (MilliporeSigma™ pET-41a(+) DNA Vector, Catalog No. 70-556-3) by Suzhou Junji Biotechnology Co., Ltd. The corresponding primers were synthesized by Suzhou Junji Biotechnology Co., Ltd. according to the sequences of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID NO. 10:

[0046] FucTd-2C

[0047] C169S Forward: 5'-CCGAACCTGTCCGCGGTGGTG-3'

[0048] C169S Reverse: 5'-CACCACCGCGGACAGGTTCGG-3'

[0049] C238S Forward: 5'-TTTAACCTGTCCTTTTGAAAC-3'

[0050] C238S Reverse: 5'-GTTTTCAAAGGACAGGTTAAA-3'

[0051] FucTd_C406

[0052] 5'-CGTAAACTGCCATCATCACC-3'

[0053] 5'-GGTGATGATGGCAGTTTACG-3'

[0054] FucTd_C412

[0055] 5'-CCATCATCACTGCTAATAAAG-3'

[0056] 5'-CTTTATTAGCAGTGATGATGG-3'

[0057] FucTd-2C_C406

[0058] Forward: 5'-CATATGTTTCAGCCGCTGCTG-3'

[0059] Reverse: 5'-GTTTACGCGTAAGTCATCGTAATTG-3'

[0060] First, FucTd-2C, FucTd_C406, and FucTd_C412 were prepared using the recombinant truncation (FucTd) SEQ ID NO. 1 as a DNA template; when preparing FucTd-2C_C406, FucTd-2C SEQ ID NO. 3 was used as a DNA template.

[0061] The PCR reaction conditions are as follows:

[0062] To a 50 μl PCR reaction mixture, 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), 1 μl of 50× Diversify dNTP Mix (10 mM each of dATP, dCTP, dGTP, and dTTP), 10 μM each of two primers, 50 ng of DNA template, 20 units of Titanium Taq, and bring the final volume to 50 μl with PCR-grade water. All reagents are from Clontech, Cat. No. 639141.

[0063] After thoroughly mixing and centrifuging the reaction components, the PCR tubes were placed in a PCR thermocycler with the following PCR reaction parameters: 94°C for 30 seconds; 25 cycles of (94°C for 30 seconds, 68°C for 1 minute); 68°C for 1 minute; and 4°C incubation.

[0064] The PCR product was subjected to agarose gel electrophoresis, excised, and purified. Then, in a 10-μl reaction volume, 1 μl (50 ng) of T-vector (pMD™19-T Vector Cloning Kit, Takara, Cat. No. 6013) was added to an equal number of PCR products. 1 μl of 10× Buffer containing ATP and the appropriate amount of T4 DNA ligase were added, and the volume was made up to 10 μl with ddH₂O. Ligation was performed overnight at 16°C. The ligated product was transformed into E. coli as follows:

[0065] (1) Preheat LB plate containing Amp, X-Gal, and IPTG to 37°C.

[0066] (2) Add 10 μl of ligation product to 100 μl of competent cells and incubate on ice for 30 min.

[0067] (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.

[0068] (4) Add 300 μL of SOC medium to the centrifuge tube, mix well with a pipette tip, and shake gently at 37°C and 150 rpm for 60 min.

[0069] (5) 200 μl of the transformed bacterial solution was evenly spread on an LB plate containing 50 mg / ml Amp, 20 mg / ml X-gal, and 200 mg / ml IPTG. The plate was incubated at 37°C overnight. White colonies were selected and sequenced by Suzhou Junji Biotechnology Co., Ltd. Based on the sequencing results, the correct mutant sequence was selected and cloned into pET41a (MilliporeSigma™ pET-41a(+) DNA Vector, catalog number: 70-556-3).

[0070] 2. Expression of Mutants

[0071] The expression of Helicobacter pylori α-1,3-fucosyltransferase mutants is briefly described as follows

[0072] (1) Add 2 μl of plasmid to 100 μl of BL21(DE3) competent cells (ThermoFisher, catalog number: EC0114), mix immediately, and place on ice for 30 min.

[0073] (2) Heat shock at 42°C for 90 s, followed by rapid ice bath for 2 min.

[0074] (3) Add 500 μL LB medium and incubate at 37°C with shaking at 200 rpm for 60 min.

[0075] (4) Centrifuge at 6000 rpm for 1 min, discard most of the supernatant, and retain about 100-150 μL. Resuspend the cells and spread them on LB plates containing Amp. Incubate at 37°C overnight.

[0076] (5) Small-scale expression: Pick one single clone and place it in 1 mL of Amp-resistant LB medium. Culture it at 37°C, 220 rpm, and shake it for about 5 hours. Add 2.5 mL of LB liquid medium containing Amp to the tube in the previous step and culture it at 37°C, 220 rpm, and shake it overnight.

[0077] (6) Transfer the overnight culture to 20 ml of LB medium containing Amp at a ratio of 1:50 and incubate at 37°C, 220 rpm until OD600 = 0.6 (approximately 3 h). Add IPTG to a final concentration of 0.5 mM and incubate at 20°C, 220 rpm for 16 h.

[0078] (7) Measure the OD600 of the culture medium, take 10OD of the bacterial culture, centrifuge at 10,000 rpm for 2 minutes, and remove the supernatant.

[0079] (8) Resuspend the cells in 1 mL of lysis buffer (10 mM Tris-HCl, pH 8.0) and sonicate on ice for lysis. The sonication conditions were: 130 W, 4 min, on 3 s, off 3 s.

[0080] (9) After sonication, the lysate was centrifuged at 12,000 rpm for 10 min to obtain the supernatant; the supernatant was ultracentrifuged at 125,000 g at 4°C.

[0081] The expression and purification yields of the recombinant truncated form (FucTd) and various mutants of Helicobacter pylori α-1,3-fucosyltransferase are shown in the purification yield column of Table 1.

[0082] Table 1. Expression, purification, and enzyme activity comparison of Helicobacter pylori α-1,3-fucosyltransferase mutants

[0083]

[0084] Under identical induction conditions, the purified yields of the cysteine-deleted mutants FucT-2C and FucTd-2C_C406 were not significantly different from those of the recombinant truncated FucTd (12 mg / L vs 8 mg / L vs 10 mg / L), indicating that the two cysteines (C169 and C238) in the H. pylori α-1,3-fucosyltransferase do not participate in and maintain protein structure through disulfide bonds. FucTd_C406 and FucTd_C412 also did not form multimers or reduce yields due to the insertion of the additional cysteines, suggesting that C406 and C412 do not form disulfide bonds with C169 and C238, and that C169 and C238 are likely not exposed on the protein surface. Surprisingly, FucTd_C406 and FucTd_C412 not only showed no decrease in yield, but actually increased their yields by 3- to 5-fold (50 mg / L vs 30 mg / L). This suggests that the H. pylori α-1,3-fucosyltransferase may form dimers, increasing their stability; the additional cysteine ​​residues may further reinforce the homodimer formation. This was confirmed by SDS-PAGE and size exclusion chromatography (SEC) analysis. As shown in Figure 2, A shows that FucTd and all mutants appear as monomers on reducing SDS-PAGE. B shows that FucTd and FucTd-2C remain monomers on non-reducing SDS-PAGE, demonstrating that the cysteines at positions 169 and 238 are not involved in dimer formation. However, the other mutants, FucTd_C406, FucTd_C412, and FucTd-2C_C406, all form dimers, mediated by factors such as disulfide bonds. This result suggests that the cysteines at positions 406 and 412 are involved in dimer formation. C shows a molecular size comparison of FucTd-2C and FucTd_C406 using size exclusion chromatography (SEC). Both exhibit single peaks with similar column retention times, and their molecular weights are approximately 100 kD. This indicates that despite differences in SDS-PAGE, both dimers form under native, non-denaturing conditions.

[0085] 3. Enzyme Protein Purification

[0086] Following the above-mentioned expression induction conditions, the culture volume was expanded to 100 ml, and the supernatant of the sonicated lysate was obtained. The supernatant was applied to a HiTrap chelating HP column according to the manufacturer's manual and eluted with 20 mM imidazole. The eluate was pooled and dialyzed to 50 mM Tris buffer (pH 8.0). The eluate was then further purified by gel filtration chromatography (Superdex 200, GE Healthcare) to obtain a highly pure protein with a homogeneity exceeding 98%. Protein concentration was determined using a Bio-Rad protein assay kit based on the Bradford method, using bovine serum albumin as a standard.

[0087] Example 2. Enzyme Activity Analysis of Recombinant Truncated and Mutant α-1,3-Fucosyltransferases from Helicobacter pylori Using the GDP-Glo™ Glycosyltransferase Assay

[0088] The enzymatic activity of recombinant truncated and mutant α-1,3-fucosyltransferases from Helicobacter pylori was assayed using a Promega assay (schematic diagram shown in Figure 3). Highly purified GDP-fucose (GDP-Fucose) was used as the donor substrate, and fetuin (fetuin) was used as the acceptor substrate. Enzyme activity was measured according to the Promega GDP-Glo™ Glycosyltransferase Assay manual. 100 µM ultrapure GDP-fucose (Promega Cat. #VA1097) was used as the donor substrate, and 40 µM fetuin (Promega Cat. #V4961) was used as the acceptor substrate. Serial dilutions of purified fucosyltransferase mutants (0 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, and 100 ng) were added. All reactions were performed in 25 µL volumes in white 96-well plates. Incubate at room temperature for 60 minutes, then place in a GloMax® 96-well microplate luminometer (Cat# E6501). Read the results at room temperature. A graph is plotted with the mass of enzyme in the reaction system on the horizontal axis and the data read on the luminometer on the vertical axis, with the slope used as an indicator of activity. The results are summarized in the Enzyme Activity Ratio (GDP Glo Glycosyltransferase Assay) column in Table 1. There are no significant differences in enzymatic activity between the mutants and the recombinant truncation (FucTd), suggesting that the wild-type C169 and C238 within the sequence and the additionally introduced C406 and C412 are not directly involved in the formation of the enzyme active center.

[0089] Example 3. Comparison of the Enzyme Activities of Recombinant Truncated and Mutant α-1,3-Fucosyltransferases from Helicobacter pylori Using Flow Cytometry

[0090] The principle is shown in Figure 4. Eukaryotic cell membranes are rich in glycoprotein molecules that can serve as receptor substrates for fucosyltransferases. In this application, NK-92 cells serve as receptor substrates. GDP-fucose-labeled Herceptin (trastuzumab for injection) is used as the donor substrate. The mean fluorescence intensity (MFI) of flow cytometry can be used to compare the amount of target molecule on the cell membrane under identical conditions and can serve as an indicator of the enzyme reaction product.

[0091] NK-92 (natural killer cells from human malignant non-Hodgkin's lymphoma patients) were purchased from Wuhan Pronocell Life Science Co., Ltd. The preparation of Herceptin and the experimental process of GDP-fucose labeling of Herceptin are shown in Example 4. 6 100 μg / mL of recombinant truncated and mutant versions of Helicobacter pylori α-1,3-fucosyltransferase and 150 μg / mL of GDP-fucose-labeled Herceptin were added to 1 mL of NK-92 cells and incubated at room temperature for 30 minutes. The cells were washed with PBS, pH 7.4, centrifuged at 500 × g, and the supernatant removed. NK-92 cells were resuspended in PBS, pH 7.4, and incubated with anti-hIgG-Biotin antibody (Beijing Biolab 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 analyzed by flow cytometry (Beijing Layer Biotechnology Co., Ltd., product number MateCyte) and analyzed with NovoExpress software. Under the same experimental conditions, the mean fluorescence intensity (MFI) can reflect the amount of target protein on the cell membrane surface. Comparison of the mean fluorescence intensity (MFI) of NK-92 cells treated with a recombinant truncated form and a mutant form of Helicobacter pylori α-1,3-fucosyltransferase revealed no significant difference in MFI between the recombinant form and the mutant form, as summarized in the Enzyme Activity Ratio (Enzyme-Mediated Antibody-Cell Coupling / FACS) column in Table 1.

[0092] Example 4. Preparation of GDP-Fucose Labeled Herceptin

[0093] Herceptin (trastuzumab for injection) was designed based on the sequence published in the literature and the plasmid DNA was synthesized by Suzhou Junji Biotechnology Co., Ltd. and cloned into the vector pRM293 (pRM293 was obtained by modifying the plasmid pTT5. For details, see Shi C. Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 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-purify the plasmid using Mabselect sure (Protein A, GE healthcare).

[0094] GDP-Fucose labeling of Herceptin involves two steps:

[0095] 1. The NHS ester-activated crosslinker and the labeled compound react with primary amines on Herceptin 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 primary amines on the Herceptin protein to produce TCO-PEG4-Herceptin. The specific method and process are as follows:

[0096] 400 μg of purified Herceptin protein was reacted with 150 μg of TCO-PEG4-NHS (Shanghai Perri 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, the reaction was terminated by adding 5 μmol Tris buffer and incubated at room temperature for 5 minutes. The reaction product was added to a PD SpinTrap G-25 desalting column (Cytiva, product number 28918004) and centrifuged at 800 × g to remove unreacted and generated small molecules to obtain pure TCO-PEG4-Herceptin.

[0097] 2. Based on the reverse-demand Diels-Alder cycloaddition reaction between trans-cyclooctene and tetrazine, a dihydropyridazine bond is formed between TCO (trans-cyclooctene) and Tz (tetrazine). Using this principle, TCO-PEG4-Herceptin reacts with the GDP-fucose derivative GDP-Fucose-Triazole-PEG4-Tz to yield GDP-Fucose-Triazole-PEG4-PEG4-Herceptin, i.e., GDP-fucose-labeled Herceptin. The details are as follows:

[0098] The TCO-PEG4-Herceptin obtained in the previous step was further reacted with 30 μg of GDP-Fucose-Triazole-PEG4-Tz (synthesized by Yan Tang 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) and centrifuged at 800×g to remove unreacted and small molecules generated by the reaction to obtain pure GDP-fucose-labeled Herceptin.

[0099] According to MALDI-TOF analysis, labeled Herceptin carries approximately 6 to 10 GDP-fucose derivatives. Henceforth, unless otherwise stated, Herceptin is referred to as GDP-fucose-conjugated Herceptin.

[0100] Example 5. Magnetic Bead-coupled Fucosyltransferase

[0101] Helicobacter pylori α-1,3-fucosyltransferase mutants FucTd_C406, FucTd_C412, and FucTd-2C_C412 all have an additional free cysteine ​​residue. They were coupled to the magnetic bead surface using a cysteine-specific site-specific coupling technique. Since FucTd_2C lacks free Cys and therefore lacks Cys-mediated coupling ability, it was not selected for coupling experiments in this example. Two methods are described, including:

[0102] 1. A stable thioether bond was formed between the cysteine ​​sulfhydryl group and biotin via maleimide (NHS). The high affinity of biotin for streptavidin was then utilized to couple the mutant to streptavidin-coated magnetic beads. The method was briefly described as follows: FucTd_C406, FucTd_C412, and FucTd-2C_C406 proteins were dissolved in PBS buffer (pH 7.4) to a final concentration of 2 mg / mL. Freshly prepared BMCC-biotin (1-Biotinamido-4-4´-[maleimidomethyl]cyclohexanecarboxamido-butane, ThermoFisher, Cat. No. 21900) was added to 2.1 mg of BMCC-biotin to prepare an 8 mM stock solution. 17 µL of BMCC-biotin was added to 1 mL of protein solution. The reaction was allowed to react at room temperature for 2 hours. The reaction product was applied to a PD SpinTrap G-25 desalting column (Cytiva, Catalog No. 28918004) and centrifuged at 800 × g to remove unreacted proteins and small molecules generated by the reaction, thereby obtaining the biotinylated proteins. Biotinylation levels were measured using the Pierce™ Fluorescent Biotin Quantitation Kit (ThermoFisher, Catalog No. 46610), demonstrating similar biotinylation levels among the three proteins. 10 µg of biotinylated protein was mixed with 100 µL of Streptavidin Dynabeads™ M-280 (ThermoFisher, Catalog No. 11205D) in PBS buffer at room temperature and incubated for 30 minutes. The proteins coupled to the beads were magnetically separated and washed 4–5 times with PBS containing 0.1% BSA before use.

[0103] 2. Utilize the free cysteine ​​residues of the mutant to directly couple to the epoxy groups on epoxy resin magnetic beads, forming a covalent bond. The method is briefly described as follows: Dynabeads™ M-270 epoxy resin microbeads (ThermoFisher, Cat. No. 14301) are single-particle, 2.8-µm superparamagnetic polystyrene microbeads coated with a hydrophilic polyether crosslinking layer. The epoxy functional groups on the beads form disulfide bonds with the free cysteine ​​residues of the protein via sulfhydryl groups. The coupling method is briefly described as follows: Weigh 5 mg of lyophilized magnetic beads (3.3 × 10 8), wash, and resuspend the beads in 100 μL PBS (pH 7.4). Add 100 μL of the target protein, mix thoroughly, and then add 100 μL 3M ammonium sulfate. Repeat mixing and incubate at 37°C for 16-24 hours. Wash the beads four times with PBS (pH 7.4) and resuspend them in 165 μL PBS (pH 7.4) before use.

[0104] Example 6. GDP-Glo™ Glycosyltransferase Assay Comparison of Enzyme Activity of Magnetic Bead-Conjugated and Free Fucosyltransferases;

[0105] Protein concentration was determined using a Bio-Rad protein assay kit based on the Bradford assay, using bovine serum albumin as a standard. Free protein and magnetic bead-coupled protein were adjusted to 50 µg / mL. 100 µM ultrapure GDP-fucose (Promega Cat. #VA1097) was used as the donor substrate, and 40 µM fetuin (Promega Cat. #V4961) was used as the acceptor substrate. Fucosyltransferase mutants and their magnetic bead conjugates were added to a 25 µL reaction system in a serial dilution series of 0 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, and 100 ng. All enzyme reactions were performed in white 96-well plates. Incubate at room temperature for 60 minutes, place in a GloMax® 96 microplate luminometer (Cat# E6501), and read at room temperature. Plot the data with the mass of enzyme in the reaction system on the horizontal axis and the data read on the luminometer on the vertical axis, and use the slope as the activity indicator.

[0106] As shown in Figure 5 and Table 2, the enzyme activities of the three mutants FucTd_C406, FucTd_C412, and FucTd-2C_C406 and their magnetic bead conjugates were compared. Magnetic bead conjugation did not affect the enzyme activity, and the activities of all mutants tended to be slightly higher than those of the corresponding free proteases, suggesting that the solid phase surface can improve the stability of the enzyme.

[0107] Table 2. Comparison of enzyme activities of three mutants and their magnetic bead conjugates

[0108]

[0109] Example 7. Comparison of the enzyme activities of magnetic bead-coupled and free fucosyltransferase mutants by flow cytometry.

[0110] The mean fluorescence intensity (MFI) of flow cytometry can be used to compare the amount of target molecules on the cell membrane under the same conditions and can be used as an indicator of the enzyme reaction product. Similar to Example 3, NK-92 cells were used as the receptor substrate and Herceptin (trastuzumab for injection) labeled with GDP-fucose was used as the donor substrate. 6 NK-92 cells were added with Helicobacter pylori α-1,3-fucosyltransferase mutants (FucTd_C406, FucTd_C412, and FucTd-2C_C406) or their magnetic bead conjugates at a final concentration of 50 μg / mL, along with GDP-fucose-labeled Herceptin at a final concentration of 100 μg / mL. The cells were incubated at room temperature for 30 minutes. The cells were washed with PBS, pH 7.4, and centrifuged at 500 × g, and the supernatant was removed. NK-92 cells were resuspended in PBS (pH 7.4) and incubated with anti-hIgG-Biotin antibody (Beijing Biolab Technology Co., Ltd., product number F030822, 1:200 dilution) at 4°C for 30 minutes. After washing once with PBS, cells were stained with Streptavidin-PE (BioLegend, Inc., product number 405204, 1:200 dilution) and incubated for 30 minutes at 4°C. After washing once with PBS, cells were analyzed by flow cytometry (Beijing Layer Biotechnology Co., Ltd., product number MateCyte) and analyzed with NovoExpress software. Under the same experimental conditions, the mean fluorescence intensity (MFI) reflects the amount of target protein on the cell membrane surface. As shown in Figure 6, Helicobacter pylori α-1,3-fucosyltransferase mutants (FucTd_C406, FucTd_C412, and FucTd-2C_C406) or their magnetic bead conjugates were able to complete the transfer of GDP-fucose (GDP-Fucose)-labeled Herceptin to NK-92 cells. There was no significant difference between the free enzyme and the magnetic bead-conjugated enzyme, and the coupling efficiency was very high, reaching 100% coupling in 30 minutes at room temperature. The MFI can indirectly reflect the amount of target protein on the cell membrane surface. Table 3 shows that magnetic bead-conjugated FucTd_C412 and FucTd-2C_C406 both increased the coupling amount, while magnetic bead-conjugated FucTd_C406 did not. In combination with the GDP-Glo™ experiment in Example 6, only the solid-phase coupling of FucTd_C406 resulted in a slight decrease in the enzyme coupling amount compared to the free enzyme. The others all showed an increase in enzyme activity or an increase in the coupling amount due to the solid-phase coupling.

[0111] Table 3. Comparison of the conjugation capacity of three mutants and their magnetic bead conjugates using mammalian cells as substrates

[0112]

[0113] Example 8. Activity Detection of Reusable Magnetic Bead-Coupled Fucosyltransferase Mutants;

[0114] As shown in Figure 7, A shows that the receptor substrate is a specific protein molecule, and B shows that the receptor substrate is a mammalian cell; both different substrates can reuse the magnetic bead-coupled fucosyltransferase to achieve the effect of coupling and modifying specific protein molecules or mammalian cells. The enzyme is site-coupled to the magnetic beads, and the catalytic reaction is completed in the solution. Then, the magnetic beads are separated and cleaned from the reactants by magnetic separation. It is easy to recycle and can be reused repeatedly. In order to detect the stability of the enzyme during repeated use, the following experiments were performed:

[0115] Protein concentration was determined using a Bio-Rad protein assay kit based on the Bradford method, using bovine serum albumin as a standard. Magnetic bead-coupled FucTd_C406, FucTd_C412, and FucTd-2C_C406 were adjusted to 50 µg / mL. 100 µM ultrapure GDP-fucose (Promega Cat. #VA1097) was used as the donor substrate, and 40 µM fetuin (Promega Cat. #V4961) was used as the acceptor substrate. To a 100µL reaction system, 100ng of each of the three fucosyltransferase mutant magnetic bead conjugates was added and incubated at room temperature for 60 minutes. The centrifuge tube was placed in a DynaMag™-2 magnetic bead separation magnet and allowed to absorb for 2 minutes. The supernatant was transferred to a white 96-well plate and placed in a GloMax® 96 Microplate Luminometer (Cat# E6501). The data were read at room temperature, representing the first use of the magnetic beads. The beads were then washed three times on the magnet. The supernatant was removed, and fresh donor and acceptor substrates were added. The reaction was incubated at room temperature for 60 minutes. The supernatant was read as described above, representing the second use of the magnetic beads. This was repeated for the third, fourth, and fifth times.

[0116] As shown in Figure 8, after five repeated uses, the enzyme activity decreased by less than 4% (as shown in Table 4). After each wash, there was a small amount of magnetic bead loss, which may be the main reason for the gradual decrease in enzyme activity.

[0117] Table 4. Comparison of enzyme activity of magnetic bead conjugates of three mutants after five repeated uses

[0118] Industrial Applicability

[0119] The invention discloses a mutant of Helicobacter pylori α-1,3-fucosyltransferase. The mutant of Helicobacter pylori α-1,3-fucosyltransferase can be industrially prepared and has industrial applicability.

Claims

1. A mutant of Helicobacter pylori α-1,3-fucosyltransferase, characterized in that, The amino acid sequence of the mutant of Helicobacter pylori α-1,3-fucosyltransferase is as shown in SEQ ID NO.3, or as shown in SEQ ID NO.5, or as shown in SEQ ID NO.7, or as shown in SEQ ID NO.

9.

2. A polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase according to claim 1.

3. The polynucleotide according to claim 2, wherein The sequence of the polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase with the amino acid sequence as shown in SEQ ID NO.3 is as shown in SEQ ID NO.4; or The sequence of the polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase with the amino acid sequence as shown in SEQ ID NO.5 is as shown in SEQ ID NO.6; or The sequence of the polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase with the amino acid sequence as shown in SEQ ID NO.7 is as shown in SEQ ID NO.8; or The sequence of the polynucleotide encoding the mutant of Helicobacter pylori α-1,3-fucosyltransferase with the amino acid sequence as shown in SEQ ID NO.9 is as shown in SEQ ID NO.

10.

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

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

6. A mutant of Helicobacter pylori α-1,3-fucosyltransferase according to claim 1 coupled to a solid-phase carrier.

7. The mutant of Helicobacter pylori α-1,3-fucosyltransferase conjugated with a solid phase carrier according to claim 6, characterized in that, The solid-phase carrier is a magnetic bead.

8. The preparation method of the mutant of Helicobacter pylori α-1,3-fucosyltransferase conjugated with magnetic beads according to claim 7, characterized in that, The method includes: (1) directly coupling the free sulfhydryl group on the mutant of fucosyltransferase to the surface of the magnetic bead, or, (2) performing site-directed biotinylation on the free sulfhydryl group, and then coupling it to the surface of the magnetic bead by means of the biotin-streptavidin affinity method.

9. A method for transferring a donor substrate conjugated with a biomacromolecule to a receptor substrate by repeatedly applying the mutant of Helicobacter pylori α-1,3-fucosyltransferase conjugated with a solid-phase carrier according to claim 6, characterized in that, The method includes the following steps: After completing the transfer of GDP-fucose conjugated with a biological macromolecule to the target cell, the magnetic bead is washed in a magnetic field environment. After removing the supernatant, fresh donor substrate and acceptor substrate are added, and the donor substrate conjugated with the biological macromolecule is transferred to the acceptor substrate again.

10. The method according to claim 9, wherein The biological macromolecule is an antibody.

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