A prokaryotic expression method for human fucosetransferase 8 and its product

Abstract

This invention discloses a prokaryotic expression method for fucotransferase 8 and its product. The prokaryotic expression method for fucotransferase 8 includes: constructing a truncated fucotransferase 8 to obtain Fut8ΔTM; constructing a recombinant expression plasmid; transforming the recombinant expression plasmid into an expression host bacterium; expressing and purifying the plasmid; wherein the amino acid sequence of Fut8ΔTM is shown in SEQ ID NO:1; and a prokaryotically expressed fucotransferase 8 comprising the core region amino acid sequence of fucotransferase 8, the core region amino acid sequence of which is shown in SEQ ID NO:1. This invention successfully expresses human truncated Fut8 (Fut8ΔTM) using *Escherichia coli*, enabling the rapid production of large quantities of this protein while maintaining its high biological activity. It features high expression efficiency, low levels of contaminating proteins, easy purification, and low cost.

Description

Technical Field

[0001] This invention belongs to the field of prokaryotic expression technology, specifically relating to a prokaryotic expression method for human fucosyltransferase 8 and its product. Background Technology

[0002] Fucosylation is a common post-translational modification of glycoproteins. Core fucosylation involves adding an α1-6-linked fucose to the first N-acetylglucosamine at the reducing end of the core pentasaccharide structure (Gn2Man3) of the N-glycan chain. This fucosylation reaction occurs in the Golgi apparatus. Studies have shown that core fucosylated glycoproteins are particularly abundant in brain tissue, and core fucosylation is closely related to tumor development, making it a consistently popular research topic in biology and medicine. The glycosyltransferase that catalyzes core fucosylation is fucotransferase Fut8, a type II membrane protein residing on the Golgi apparatus membrane and the only fucotransferase capable of catalyzing the formation of α1-6 fucosidic bonds.

[0003] Fut8, short for alpha-(1,6)-fucosyltransferase, was initially purified and active from pig kidneys. For a period afterward, research on Fut8 utilized naturally extracted and purified proteins from sources including human skin fibroblasts, bovine gamma globulin, and human gastric and liver cancer cells. Currently, commercially available insect cell expression of hamster / human truncated Fut8 is primarily supplied by Sino Biologicals. However, insect expression systems have low yields and are costly, resulting in a very high price for the commercially available protein, with 20 μg of protein costing as much as 2310 RMB.

[0004] Prokaryotic expression systems, such as those in *E. coli*, offer high yields and contain few interfering proteins, making them ideal for large-scale in vitro expression of recombinant Fut8 protein. To date, there have been no reports of *E. coli* expressing human Fut8, primarily because Fut8 is a Golgi-resident mammalian membrane protein with glycosylation modifications, making it prone to degradation in prokaryotic systems and difficult to express and purify. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the above-mentioned technical deficiencies, the present invention is proposed.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a prokaryotic expression method for fucosyltransferase 8 and its product.

[0008] Therefore, as one aspect of the present invention, the present invention overcomes the deficiencies of the prior art and provides a prokaryotic expression method for fucotransferase 8, which includes: constructing a truncated fucotransferase 8 to obtain Fut8ΔTM; constructing a recombinant expression plasmid; transforming the recombinant expression plasmid into an expression host bacterium, expressing and purifying it; wherein, the amino acid sequence of Fut8ΔTM is shown in SEQ ID NO:1.

[0009] As a preferred embodiment of the prokaryotic expression method of fucosyltransferase 8 according to the present invention, wherein: the fucosyltransferase 8 is human fucosyltransferase 8, and its amino acid sequence is shown in SEQ ID NO:2; the expression plasmid is pET28a, the recombinant expression plasmid is pET28a-Fut8ΔTM, and the expression host bacterium is ROSETTA.

[0010] As a preferred embodiment of the prokaryotic expression method of fucotransferase 8 according to the present invention, the expression is performed by culturing and inducing the expression host bacteria in a culture medium; the purification is performed by ultrasonically disrupting the expression host bacteria and then purifying it by nickel affinity chromatography.

[0011] In a preferred embodiment of the prokaryotic expression method of fucotransferase 8 according to the present invention, the expression involves first culturing the expression host bacteria at 37°C and 200 r / min with shaking to allow OD to reach a certain level. 600 Once the pH reaches 0.6–0.8, cool the temperature to 16°C and continue culturing. Add IPTG and induce culture at 16°C and 200 r / min.

[0012] As a preferred embodiment of the prokaryotic expression method of fucosyltransferase 8 according to the present invention, the purification involves: cleaving the expression host bacteria, resuspending it in buffer A, adding Ni-NTA agarose and incubating at 4°C for 45 min by rotation, centrifuging at low speed, washing the column with buffer A and buffer A containing a first concentration of imidazole, and eluting the protein with buffer A containing a second concentration of imidazole and buffer A containing a third concentration of imidazole; wherein, buffer A is a mixed solution comprising tris(hydroxymethyl)aminomethane, HCl, and NaCl; the first concentration is less than or equal to the second concentration, and the second concentration is less than or equal to the third concentration.

[0013] As a preferred embodiment of the prokaryotic expression method of fucosyltransferase 8 according to the present invention, the purification involves: lysing the expression host bacteria, resuspending it in buffer A, adding Ni-NTA agarose and incubating at 4°C for 45 min by rotation, centrifuging at low speed, washing the column with buffer A and buffer A containing 10-30 mM imidazole respectively, and eluting the protein with buffer A containing 45-85 mM imidazole and buffer A containing 150-350 mM imidazole; wherein, buffer A is a mixed solution of Tris / HCl and NaCl at pH 8.0.

[0014] In another aspect, the present invention overcomes the shortcomings of the prior art by providing a prokaryotically expressed fucotransferase 8, which includes the amino acid sequence of the core region of fucotransferase 8, as shown in SEQ ID NO:1.

[0015] As a preferred embodiment of the prokaryotically expressed fucotransferase 8 of the present invention, wherein: the protein catalytic substrate of the fucotransferase 8 includes one or more of Fmoc-Asn-Gn2Man3Gn2, Fmoc-Asn-Gn2Man3Gn and Fmoc-Asn-Gn2Man5, and the oligosaccharide donor of the fucotransferase 8 is GDP-L-Fucose.

[0016] As a preferred embodiment of the prokaryotically expressed fucosyltransferase 8 described in this invention, the enzyme activity is 36 IU.

[0017] As a preferred embodiment of the prokaryotic expression of fucosyltransferase 8 described in this invention, the catalysis of Fmoc-Asn-Gn2Man3Gn achieves a conversion rate of 100%.

[0018] The beneficial effects of this invention are:

[0019] This invention successfully expresses human truncated Fut8 (Fut8ΔTM) using Escherichia coli, which can produce large quantities of the protein in a short time while maintaining its high biological activity. It features high expression efficiency, few contaminating proteins, easy purification, and low cost and convenience. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0021] Figure 1Immunoblot detection of Fut8ΔTM protein (Figure A): 1: Cell lysis buffer before induction, 2: Cell lysis buffer after induction, Antibody: anti-His; Protein electrophoresis SDS-PAGE Coomassie Brilliant Blue staining (Figure B): 1: Blue Plus II Marker, 2: Cell lysis buffer before induction, 3: Cell lysis buffer after induction, 4: Purified Fut8ΔTM protein.

[0022] Figure 2 Figure A shows the reaction process of Fut8 protein in vivo and the HPLC detection of the activity of purified Fut8ΔTM protein in vitro. Figure B shows: 1: Fut8ΔTM reaction system and substrate Fmoc-Asn-Gn2Man3Gn2 standard sample mixture; 2: Fut8ΔTM reaction system; 3: substrate Fmoc-Asn-Gn2Man3Gn2 standard sample.

[0023] Figure 3 The images show the specific HPLC detection of the Fut8 protein donor: 1: Fut8ΔTM reacted with GDP-Mannose for 2 hours (positive control); 2: Fut8ΔTM reacted with GDP-L-Fucose for 2 hours; 3: substrate Fmoc-Asn-Gn2Man3Gn2 standard sample.

[0024] Figure 4 Figure A shows the substrate-specific HPLC detection of Fut8 protein. The reaction of Fut8 with Fmoc-Asn-Gn2Man3Gn (Figure A): 1: a mixture of Fmoc-Asn-Gn2Man3 and Fmoc-Asn-Gn2Man3Gn; 2: the reaction system of the mixture of Fmoc-Asn-Gn2Man3 and Fmoc-Asn-Gn2Man3Gn with Fut8; and the reaction of Fut8 with Fmoc-Asn-Gn2Man5 (Figure B). Detailed Implementation

[0025] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0027] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0028] The reagents used in this example were as follows: restriction endonucleases, Taq DNA polymerase, and ligases were purchased from TaKaRa (Japan); the oligosaccharide substrate Fmoc-Asn-Gn2Man3Gn2 was a gift from the National Institute of Advanced Industrial Science and Technology (AIST) of Japan; gel extraction, PCR product purification, and plasmid extraction kits and IPTG were purchased from Shanghai Sangon Biotech Co., Ltd.; GDP-L-Fucose was purchased from Qingdao Shuge Biotechnology Co., Ltd.; and all other reagents were purchased from Sinopharm. Primer synthesis and sequencing were performed at BGI Genomics.

[0029] Example 1: Prokaryotic expression and purification of human Fut8ΔTM

[0030] Based on protein structure analysis from the UniPort database, the N-terminal transmembrane domain of Fut8 was truncated (Fut8ΔTM, aa68-545), constructing the prokaryotic expression vector pET28a-Fut8ΔTM. The recombinant prokaryotic expression plasmid was transformed into ROSETTA prokaryotic expression host bacteria and plated on LB + kanamycin + chloramphenicol (34 μg / mL) plates. The next day, a single colony was picked from the transformation plate and inoculated into 5 mL of LB + kanamycin + chloramphenicol liquid medium, and cultured overnight at 37°C with shaking. 2 mL of the overnight culture was inoculated into 200 mL of TB + kanamycin + chloramphenicol liquid medium and cultured at 37°C with shaking at 200 rpm for 3 h to allow OD to adjust. 600 After reaching a pH of 0.6-0.8, the culture was cooled to 16℃ and incubated for another hour. Then, IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 200 rpm for 20 hours. The bacterial cells were collected by centrifugation and resuspended in 20 mL of buffer A (25 mM Tris / HCl, 150 mM NaCl, pH 8.0). The pET28a-Fut8ΔTM recombinant bacteria were sonicated and centrifuged at 9000 rpm for 60 minutes. The precipitate was discarded, and the supernatant was collected for purification.

[0031] Fut8ΔTM Protein Purification 1: Add 1 mL of Ni-NTA Agarose to 20 mL of supernatant and incubate at 4°C for 45 min by rotation. Collect the gel by low-speed centrifugation for column packing. Wash the column with 10 mL of buffer A and 10 mL of buffer A containing 20 mM imidazole, respectively. Elute the protein with 6 mL of buffer A containing 60 mM imidazole and 5 mL of buffer A containing 250 mM imidazole, collecting 1 mL of eluent from each centrifuge tube. Perform SDS-PAGE electrophoresis on the samples. Figure 1B indicates that the above-mentioned Fut8ΔTM was successfully purified.

[0032] Fut8ΔTM Protein Purification 2: Add 1 mL of Ni-NTA Agarose to 20 mL of supernatant and incubate at 4°C for 45 min by rotation. Collect the gel by low-speed centrifugation for column packing. Wash the column with 10 mL of buffer A and 10 mL of buffer A containing 20 mM imidazole, respectively. Elute the protein with 10 mL of buffer A containing 250 mM imidazole, collecting 1 mL of eluent from each centrifuge tube. SDS-PAGE electrophoresis analysis showed the presence of contaminating proteins, indicating unsatisfactory purification results.

[0033] Example 2: In vitro activity assay of Fut8ΔTM

[0034] The standard enzyme activity assay system was prepared as follows (50 μL): 100 mM MES / NaOH (pH 6.0), 0.1 μM Fmoc-Asn-Gn2Man3Gn2, 0.1 mM GDP-L-Fucose, and 20 μg / mL Fut8ΔTM. The reaction system was incubated at 37 °C for 5 h. After the reaction, the system was centrifuged at 15000 r / min for 5 min, and the supernatant was collected for high-performance liquid chromatography (HPLC) analysis. The reaction system was then compared with the standard sample.

[0035] The high-performance liquid chromatography (HPLC) detection conditions were as follows: An Alliance e2695 HPLC (Waters) was used, with a 2475FLR Detector (Waters) used for fluorescence detection. An amino column (TOSHO TSKgel Amide-80 3μm 4.6×150mm) was used for detecting Fmoc-tagged sugar chains. The excitation wavelength of the fluorescence detector was 260nm, and the detection wavelength was 315nm. The elution conditions were linear gradient elution with acetonitrile (CH3CN) and ammonium acetate (NH4OAc) (Solution A: CH3CN; Solution B: 0.2M NH4OAc pH 5.0; Elution conditions: 0-35 min, 85%-55% A; 35-40 min, 55%-20% A; 40-45 min, 20%-84% A; 45-50 min, 85% A; Flow rate: 1 mL / min) to separate the substrate and product.

[0036] The results are as follows Figure 2 B shows that the product peak obtained from the reaction system did not elute at the same time as the substrate Fmoc-Asn-Gn2Man3Gn2, and the mixture of product and substrate showed two elution peaks at different times, indicating that Fut8ΔTM obtained from the E. coli expression system has catalytic activity in vitro. The yield of Fut8ΔTM was measured to be 4 mg / L.

[0037] Example 3: Enzyme activity detection of Fut8ΔTM

[0038] The enzyme activity assay system was prepared as follows (50 μL): 100 mM MES / NaOH (pH 6.0), 0.1 μM Fmoc-Asn-Gn2Man3Gn2, 0.1 mM GDP-L-Fucose, and 20 μg / mL Fut8ΔTM. The reaction system was incubated at 37 °C for 45 min. After the reaction, the system was centrifuged at 15000 r / min for 5 min, and the supernatant was collected for high-performance liquid chromatography (HPLC) analysis. The reaction system was then compared with the standard sample.

[0039] The substrate conversion rate was found to be 38.75%, and the enzyme activity was 36 IU.

[0040] Example 4: Donor-specific detection of Fut8ΔTM

[0041] The donor-specific detection system was as follows (50 μL): 100 mM MES / NaOH (pH 6.0), 0.1 μM Fmoc-Asn-Gn2Man3Gn2, 10 μM GDP-Mannose, 20 μg / mL Fut8ΔTM. The reaction system was incubated at 37 °C for 5 h. After the reaction, the system was centrifuged at 15000 r / min for 5 min, and the supernatant was collected for detection by high-performance liquid chromatography (HPLC). The reaction system was then compared with standard samples.

[0042] The results are as follows Figure 3 The results showed that in the positive control system, the main peak of the Fut8ΔTM reacted with GDP-L-Fucose for 2 hours shifted backward, indicating the formation of Fmoc-Asn-Gn(Fuc)GnMan3Gn2. However, in the system of Fut8ΔTM reacting with GDP-Mannose for 2 hours, the substrate peak of Fmoc-Asn-Gn2Man3Gn2 did not shift, indicating no new product formation. These results indicate that Fut8ΔTM cannot recognize GDP-Mannose as a donor for this reaction.

[0043] Example 5: Substrate-specific detection of Fut8ΔTM

[0044] The substrate-specific detection system was as follows (50 μL): 100 mM MES / NaOH (pH 6.0), 0.1 μM Fmoc-Asn-Gn2Man3Gn (or Fmoc-Asn-Gn2Man3Gn2Gal2 or Fmoc-Asn-Gn2Man3 or Fmoc-Asn-Gn2Man5), 10 μM GDP-L-Fucose, 20 μg / mL Fut8ΔTM. The reaction system was incubated at 37 °C for 5 h.

[0045] The results are as follows Figure 4 The display shows that, for example, Figure 4 As shown in Figure A, Fut8ΔTM can recognize Fmoc-Asn-Gn2Man3Gn as equivalent to the natural substrate Fmoc-Asn-Gn2Man3Gn2 with a conversion rate of 100%; for Fmoc-Asn-Gn2Man5, the conversion rate is approximately 26% after 24 hours of incubation. Figure 4 B).

[0046] In addition, Fmoc-Asn-Gn2Man3Gn2Gal2 and Fmoc-Asn-Gn2Man3 are almost impossible to recognize.

[0047] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention. sequence list <110> Jiangnan University <120> A prokaryotic expression method for fucotransferase 8 and its product <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 545 <212> PRT <213> Artificial Sequence <400> 1 Arg Asp Asn Asp His Pro Asp His Ser Ser Arg Glu Leu Ser Lys Ile 1 5 10 15 Leu Ala Lys Leu Glu Arg Leu Lys Gln Gln Asn Glu Asp Leu Arg Arg 20 25 30 Met Ala Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly Pro 35 40 45 Ala Ile Gly Arg Val Arg Val Leu Glu Glu Gln Leu Val Lys Ala Lys 50 55 60 Glu Gln Ile Glu Asn Tyr Lys Lys Gln Thr Arg Asn Gly Leu Gly Lys 65 70 75 80 Asp His Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu Leu 85 90 95 Trp Phe Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Asn Leu Glu Gly 100 105 110 Asn Glu Leu Gln Arg His Ala Asp Glu Phe Leu Leu Asp Leu Gly His 115 120 125 His Glu Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp 130 135 140 Gly Ala Gly Asp Trp Arg Glu Lys Glu Ala Lys Asp Leu Thr Glu Leu 145 150 155 160 Val Gln Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys 165 170 175 Ala Lys Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys 180 185 190 Gln Leu His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln 195 200 205 Arg Thr Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala Thr Gly Gly 210 215 220 Trp Glu Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser 225 230 235 240 Gly Ile Ser Thr Gly His Trp Ser Gly Glu Val Lys Asp Lys Asn Val 245 250 255 Gln Val Val Glu Leu Pro Ile Val Asp Ser Leu His Pro Arg Pro Pro 260 265 270 Tyr Leu Pro Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu Val Arg 275 280 285 Val His Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val Lys Tyr 290 295 300 Leu Ile Arg Pro Gln Pro Trp Leu Glu Lys Glu Ile Glu Glu Ala Thr 305 310 315 320 Lys Lys Leu Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg 325 330 335 Thr Asp Lys Val Gly Thr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr 340 345 350 Met Val His Val Glu Glu His Phe Gln Leu Leu Ala Arg Arg Met Gln 355 360 365 Val Asp Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro Ser Leu Leu 370 375 380 Lys Glu Ala Lys Thr Lys Tyr Pro Asn Tyr Glu Phe Ile Ser Asp Asn 385 390 395 400 Ser Ile Ser Trp Ser Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser 405 410 415 Leu Arg Gly Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe 420 425 430 Leu Val Cys Thr Phe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile 435 440 445 Met Gln Thr Leu His Pro Asp Ala Ser Ala Asn Phe His Ser Leu Asp 450 455 460 Asp Ile Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn Gln Ile Ala Ile 465 470 475 480 Tyr Ala His Gln Pro Arg Thr Ala Asp Glu Ile Pro Met Glu Pro Gly 485 490 495 Asp Ile Ile Gly Val Ala Gly Asn His Trp Asp Gly Tyr Ser Lys Gly 500 505 510 Val Asn Arg Lys Leu Gly Arg Thr Gly Leu Tyr Pro Ser Tyr Lys Val 515 520 525 Arg Glu Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Glu 530 535 540 Lys 545 <210> 2 <211> 575 <212> PRT <213> Artificial Sequence <400> 2 Met Arg Pro Trp Thr Gly Ser Trp Arg Trp Ile Met Leu Ile Leu Phe 1 5 10 15 Ala Trp Gly Thr Leu Leu Phe Tyr Ile Gly Gly His Leu Val Arg Asp 20 25 30 Asn Asp His Pro Asp His Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala 35 40 45 Lys Leu Glu Arg Leu Lys Gln Gln Asn Glu Asp Leu Arg Arg Met Ala 50 55 60 Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly Pro Ala Ile 65 70 75 80 Gly Arg Val Arg Val Leu Glu Glu Gln Leu Val Lys Ala Lys Glu Gln 85 90 95 Ile Glu Asn Tyr Lys Lys Gln Thr Arg Asn Gly Leu Gly Lys Asp His 100 105 110 Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu Leu Trp Phe 115 120 125 Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Asn Leu Glu Gly Asn Glu 130 135 140 Leu Gln Arg His Ala Asp Glu Phe Leu Leu Asp Leu Gly His His Glu 145 150 155 160 Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala 165 170 175 Gly Asp Trp Arg Glu Lys Glu Ala Lys Asp Leu Thr Glu Leu Val Gln 180 185 190 Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Lys 195 200 205 Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu 210 215 220 His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr 225 230 235 240 Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu 245 250 255 Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser Gly Ile 260 265 270 Ser Thr Gly His Trp Ser Gly Glu Val Lys Asp Lys Asn Val Gln Val 275 280 285 Val Glu Leu Pro Ile Val Asp Ser Leu His Pro Arg Pro Pro Tyr Leu 290 295 300 Pro Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu Val Arg Val His 305 310 315 320 Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile 325 330 335 Arg Pro Gln Pro Trp Leu Glu Lys Glu Ile Glu Glu Ala Thr Lys Lys 340 345 350 Leu Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg Thr Asp 355 360 365 Lys Val Gly Thr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Met Val 370 375 380 His Val Glu Glu His Phe Gln Leu Leu Ala Arg Arg Met Gln Val Asp 385 390 395 400 Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro Ser Leu Leu Lys Glu 405 410 415 Ala Lys Thr Lys Tyr Pro Asn Tyr Glu Phe Ile Ser Asp Asn Ser Ile 420 425 430 Ser Trp Ser Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg 435 440 445 Gly Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val 450 455 460 Cys Thr Phe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln 465 470 475 480 Thr Leu His Pro Asp Ala Ser Ala Asn Phe His Ser Leu Asp Asp Ile 485 490 495 Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn Gln Ile Ala Ile Tyr Ala 500 505 510 His Gln Pro Arg Thr Ala Asp Glu Ile Pro Met Glu Pro Gly Asp Ile 515 520 525 Ile Gly Val Ala Gly Asn His Trp Asp Gly Tyr Ser Lys Gly Val Asn 530 535 540 Arg Lys Leu Gly Arg Thr Gly Leu Tyr Pro Ser Tyr Lys Val Arg Glu 545 550 555 560 Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Glu Lys 565 570 575

Claims

1. A prokaryotic expression method for fucotransferase 8, characterized in that: The process includes: constructing a truncated fucosyltransferase 8 to obtain Fut8ΔTM; constructing a recombinant expression plasmid; transforming the recombinant expression plasmid into an expression host bacterium, expressing and purifying it; wherein the amino acid sequence of Fut8ΔTM is positions 68-545 of sequence SEQ ID NO:2; the expression plasmid is pET28a, the recombinant expression plasmid is pET28a-Fut8ΔTM, and the expression host bacterium is ROSETTA; The expression process involves first culturing the host bacteria at 37°C and 200 rpm with shaking until the OD600 reaches 0.6–0.8, then cooling to 16°C and continuing the culture. IPTG is then added, and the culture is induced at 16°C and 200 rpm. The purification process involves lysing the host bacteria, resuspending it in buffer A, adding Ni-NTA amino acid, incubating at 4°C for 45 min, centrifuging at low speed, washing the column with buffer A and buffer A containing a first concentration of imidazole, and eluting the protein with buffer A containing a second concentration of imidazole and buffer A containing a third concentration of imidazole. Buffer A is a mixed solution comprising tris(hydroxymethyl)aminomethane, HCl, and NaCl; the first concentration is less than or equal to the second concentration, and the second concentration is less than or equal to the third concentration. The expression refers to culturing and inducing the expression host bacteria in a culture medium; the purification refers to purifying the expression host bacteria by ultrasonic disruption followed by nickel affinity chromatography. The purification process involves lysing the expression host bacteria, resuspending it in buffer A, adding Ni-NTA agarose, incubating at 4°C for 45 min by rotation, centrifuging at low speed, washing the column with buffer A and buffer A containing 10–30 mM imidazole, and eluting the protein with buffer A containing 45–85 mM imidazole and buffer A containing 150–350 mM imidazole; wherein, buffer A is a mixed solution of Tris / HCl and NaCl at pH 8.

0.

2. A prokaryotically expressed fucotransferase 8, characterized in that: The 8-amino acid sequence of the fucotransferase is shown at positions 68-545 of sequence SEQ ID NO:

2.

3. The fucotransferase 8 as described in claim 2, characterized in that: The protein catalytic substrate of the fucosyltransferase 8 includes one or more of Fmoc-Asn-Gn2Man3Gn2, Fmoc-Asn-Gn2Man3Gn, and Fmoc-Asn-Gn2Man5, and the oligosaccharide donor of the fucosyltransferase 8 is GDP-L-Fucose.

4. The fucotransferase 8 as described in claim 2, characterized in that: The enzyme activity was 36 IU.

5. The prokaryotic expression method of fucotransferase 8 as described in claim 2, characterized in that: The catalytic conversion of Fmoc-Asn-Gn2Man3Gn was 100%.

Images