KR100481910B1 - Fusion protein producing galactose α-1,3-galactose-β-1,4-N-acetyl glucosamine - Google Patents

Abstract

The present invention relates to fusion proteins having α-1,3-galactosyltransferase and β-1,4-galactosyltransferase-1 activity, and more particularly, α-1,3-galactosyl A fusion protein comprising a transferase and β-1,4-galactosyltransferase-1, a gene encoding the protein, a method for mass-producing the fusion protein, and galactose-β-1 using the fusion protein, 4- N -acetylglucosamine (Galactose-β-1,4- N- acetylglucosamine) and galactose-α-1,3-galactose-β-1,4- N -acetylglucosamine (Galactose-α-1,3-galactose It relates to a method for producing a functional complex sugar having a -β-1,4- N -acetylglucosamine) structure. The fusion protein of the present invention simultaneously has α-1,3-galactosyltransferase and β-1,4-galactosyltransferase-1 activity, and thus is important for the production of trisaccharides, which are important as immunosuppressive agents in heterologous tissue transplantation. It can be usefully used.

Description

Fusion protein producing galactose α-1,3-galactose-β-1,4-N-acetyl glucosamine {1,3-galactose-β-1,4-N-acetylglucosamine}

The present invention relates to a recombinant protein having both α-1,3-galactosyltransferase and β-1,4-galactosyltransferase activity. More specifically, α-1,3-galactosyltransferase A fusion protein comprising a lase and a β-1,4-galactosyltransferase, a gene encoding the fusion protein, a method for mass production of the fusion protein, and a method for producing a functional sugar using the protein .

Complex sugars present on the surface of the cell play an important role such as mutual recognition and mutual adhesion between cells and cells (Galili, 1971) by touching the cell's surroundings first (Mary et al., 1991). Tissue-specific expression is regulated throughout the life cycle of life, from development, differentiation and aging (Feizi ., 1985; Barry , 1993). In addition, different types of complex sugars appear in different tissues of the species (Masahiro et al ., 2000).

Various types of complex sugars are synthesized by glycosyltransferases. One of the glycotransferases, galactosyltransferase, is a donor substrate UDP-Gal and a receptor substrate N -acetylglucosamine (Glc N Β-1,4-galactosyltransferase which binds to β-1,4 linkage to Ac) and α-1,3- which delivers UDP-Gal as α-1,3 linkage to other receptor substrates Divided into galactosyltransferases. Galactose-α-1,3-galactose-β-1,4, a trisaccharide with α-galactose epitopes synthesized by β-1,4-galactosyltransferase and α-1,3-galactosyltransferase N -acetylglucosamine (Gal-α-1,3-Gal-β-1,4-Glc N Ac) is an immunosuppressive inhibitor in heterologous tissue transplantation (Uri et al ., 1987; Good et al. , 1992; Galili et al ., 1993; Oriol et al., 1993; Sandrin et al ., 1993; Francisca et al ., 1994; Paker et al ., 1994; Uri et al ., 1996; McMorrow et al ., 1997) It can be utilized as an indicator for labeling cancer cells.

However, β-1,4-galactosyltransferase and α-1,3-galactosyltransferase are separately added to the trisaccharide (Gal-α-1,3-Gal-β-1,4-Glc N When synthesizing Ac), it is troublesome to synthesize disaccharide (Gal-β-1,4-Glc N Ac) first by using monosaccharide as a substrate, and to obtain this saccharide purely and then perform the second enzymatic reaction again. . In addition, there is no recombinant enzyme yet to show the activity of the two enzymes at the same time.

In this regard, the present inventors produced a fusion protein by linking α-1,3-galactosyltransferase and β-1,4-galactosyltransferase in order to mass synthesize trisaccharides, which are important as immunosuppressive agents in heterologous tissue transplantation. In addition, the method for mass production of the fusion protein and the trisaccharide galactose-α-1,3-galactose-β-1,4- N -acetylglucosamine (Gal-α-1) even in one reaction using the fusion protein The present invention was completed by revealing the possibility of synthesizing, 3-Gal-β-1,4-Glc N Ac).

An object of the present invention is a fusion protein fused with β-1,4-galactosyltransferase and α-1,3-galactosyltransferase, a gene encoding the fusion protein, a method for mass production of the fusion protein And it provides a method for producing a functional complex saccharide using the fusion protein.

In order to achieve the above object, the present invention provides a fusion protein comprising α-1,3-galactosyltransferase and β-1,4-galactosyltransferase.

The present invention also provides a gene encoding the fusion protein.

The present invention also provides a method for producing the fusion protein.

In addition, the present invention provides a functional complex saccharide having a galactose-β-1,4- N -acetylglucosamine and galactose-α-1,3 galactose-β-1,4- N -acetylglucosamine structure using the fusion protein. It provides a method of manufacturing.

Hereinafter, the present invention will be described in detail.

The present invention provides a fusion protein comprising α-1,3-galactosyltransferase and β-1,4-galactosyltransferase.

The α-1,3-galactosyltransferase is preferably one in which the amino terminus corresponding to the cytoplasmic site and the transmembrane site of the α-1,3-galactosyltransferase is removed. Specifically, SEQ ID NO: 2 More preferably having an amino acid sequence of Nos. 63 to 414.

In addition, the β-1,4- galactosyl transferase is a β-1,4- going preferably the Lactobacillus of the chamber transferase cytoplasmic membrane and the amino terminal portion corresponding to the through portions removed, specifically, the sequence it is more preferably 398 times to about 49 times the number 4 having the amino acid sequence.

In addition, the fusion protein preferably comprises a non-functional linkage at the linking site between α-1,3-galactosyltransferase and β-1,4-galactosyltransferase, and includes two amino acid sequences (Leu More preferably) -Glu). Further, it is preferable to include a histidine tag at the β-1,4-galactosyltransferase terminal. The histidine tag added at the C-terminus facilitates purification after fusion protein expression.

Most preferably, the fusion protein of the present invention has an amino acid sequence set forth in SEQ ID NO: 6 .

The fusion protein of the present invention simultaneously exhibits α-1,3-galactosyltransferase activity and β-1,4-galactosyltransferase activity, and galactose-β-1,4- N -acetylglucosamine and galactose- A functional complex saccharide having α-1,3 galactose-β-1,4- N -acetylglucosamine structure can be prepared (see FIG. 5 ).

In a preferred embodiment of the present invention, a fusion protein having an amino acid sequence set forth in SEQ ID NO: 6 and simultaneously exhibiting α-1,3-galactosyltransferase activity and β-1,4-galactosyltransferase activity is prepared. And named "α-1,3 / β-1,4-galactosyltransferase" (see FIG. 1 ).

The present invention also provides a gene encoding a fusion protein which simultaneously exhibits the α-1,3-galactosyltransferase activity and the β-1,4-galactosyltransferase activity.

Preferably, the gene has a nucleotide sequence as set forth in SEQ ID NO: 5 .

The present invention also provides a method for producing the fusion protein.

The mass production method of α-1,3 / β-1,4-galactosyltransferase of the present invention

1) preparing an expression vector comprising a histidine tag and a gene encoding a fusion protein which simultaneously exhibits α-1,3-galactosyltransferase activity and β-1,4-galactosyltransferase activity;

2) transforming the expression vector into ompT deficient E. coli;

3) culturing the transformant and inducing expression of α-1,3 / β-1,4-galactosyltransferase; And

4) purifying the α-1,3 / β-1,4-galactosyltransferase.

The ompT deficient strain E. coli BL21 (DE3) was used as a host cell, and IPTG was added to induce protein expression (see FIGS . 2 and 3 ).

In the case of purifying after expressing the fusion enzyme of the present invention, NiNTA-agarose was used (see FIG. 3 ). This is because histidine binds to metal ions. Specifically, the fusion enzyme was purified by accepting column chromatography using histidine binding resin. The histidine-binding resin may be any resin using the principle that histidine binds to the Ni metal. In a preferred embodiment of the present invention, fusion enzyme was purified by performing column chromatography using Ni-NTA resin.

However, when the transformant is expressed at 37 ° C., it is expressed in a water-soluble and insoluble state (see FIG. 2 ). Therefore, in the present invention, after culturing the transformant and inducing the expression of the fusion protein by IPTG, it is possible to obtain a fusion enzyme having high activity by using betamercaptoethanol in order to induce enzymes insoluble in water ( FIG. 4 ).

In addition, the present invention provides a galactose-β-1,4- N -acetyl using a fusion protein which simultaneously exhibits the α-1,3-galactosyltransferase activity and the β-1,4-galactosyltransferase activity. Provided is a method for preparing a functional complex saccharide having glucosamine and galactose-α-1,3-galactose-β-1,4- N -acetylglucosamine structure.

As a result of analyzing the fusion enzyme activity produced by the method of the present invention, a functional complex saccharide having a galactose-α-1,3-galactose-β-1,4- N -acetylglucosamine structure in monosaccharide N -acetylglucosamine was once It can be prepared by the reaction of, and has been confirmed to have efficient enzymatic activity (see FIG. 5 ). Therefore, the fusion protein of the present invention can be usefully used for the production of complex sugars having an immunosuppressive action during organ transplantation.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Cloning and Expression of α-1,3 / β-1,4-galactosyltransferase Gene

The inventors of the present invention have described the cDNA gene (GeneBank Accession No.:P23336) of mouse α-1,3-galactosyltransferase as described in SEQ ID NO: 1 and human β-1 described in SEQ ID NO: 3 to prepare a fusion protein. , 4-Galactosyltransferase The fusion protein was prepared using the whole cDNA gene (GeneBank Accession No: X55415).

Specifically, cDNA encoding the soluble portion from which the 62 amino acid sequence of the NH ₂ -terminal portion corresponding to the cytoplasmic site and transmembrane site of the mouse α-1,3-galactosyltransferase of SEQ ID NO: 2 is removed. Soluble portion (Arg ⁴⁹ to Ser) that removes the 48 amino acid sequence of the NH ₂ -terminal portion corresponding to the cytoplasmic and transmembrane sites of the gene and the human β-1,4-galactosyltransferase of SEQ ID NO: 4 ³⁹⁸ ) β-1,4-galactosyltransferase gene was fused 1: 1.

To this end, the inventors performed PCR using α-1,3-galactosyltransferase cDNA as a template. The design of the primers was designed based on the Genbank nucleotide sequence database (Accession number P23336) of NCBI, primers comprising the Nco I cleavage site as set out in SEQ ID NO: 7 and primers comprising the XhoI cleavage site as set out in SEQ ID NO: 8 Was used. PCR was performed by adding 5 μl of 10X reaction buffer, 2.5 μm dNTP, 0.5 μl of primer, 50 μl of each primer, 5 U of Ex Taq (Takara) to 20 ng of template DNA, and adding 50 μl of sterile distilled water. Two drops were added and then repeated 30 times at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C. PCR products were confirmed by the approximate DNA concentration by electrophoresis. The PCR product was digested with Nco I and Xho I, electrophoresed and separated from the gel to obtain the required DNA using a Geneclean kit, and purified by phenol / chloroform isoamyl alcohol to precipitate ethanol, followed by appropriate sterilization. After dissolving in water, electrophoresis confirmed the approximate DNA concentration.

In addition, in the case of β-1,4-galactosyltransferase, the N-terminus with Xho I is used to connect this vector to the α-1,3-galactosyltransferase carboxyl terminal at pET29b (Novagen, Darmstadt, Germany). The part was cut with restriction enzyme and purified in the same way.

The vector prepared above and the α-1,3-galactosyltransferase gene were reacted for 12-16 hours at 16 ° C. in order to mix and link in a 1: 1 ratio. The combined reaction product was mixed with 100 μl of competent cells (DH5α) and left for 30 minutes on ice. After heat treatment at 42 ° C. for 90 seconds, 900 μl of SOC medium was incubated at 37 ° C. for 1 hour, followed by 50 μg of kanamycin. The plate was plated in an LB culture plate containing / mL and incubated at 37 ° C for 12-16 hours.

Clones were inoculated into 5 ml of LB medium containing 50 µg / ml of kanamycin from the culture dish and incubated for 12-16 hours at 37 ° C and 250 rpm. The culture solution was centrifuged to recover the cells. Separation of the plasmids was carried out using the alkaline dissolution method. That is, the cells were precipitated by centrifugation and suspended in 200 μl of Solution I (50 mM glucose, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and 200 μl of Solution II (0.2 N NaOH, 1% SDS). Mix and dissolve the cells on ice for 5 minutes, add 200 μl of solution III (4 M potassium acetate, 2 M acetic acid, pH 4.8), mix, leave for 5 minutes on ice, and centrifuge at 4 ℃ and 12,000 rpm. Was taken. 0.5 times volume of isopropanol of the supernatant was added and centrifuged at 12,000 rpm to precipitate plasmid DNA. The precipitated DNA was washed with 100% ethanol and 70% ethanol and the ethanol was completely dried in a rotary freeze dryer. The fully dried plasmid was dissolved in 100 μl of TE containing 10 mg / ml RNase. The plasmid was digested with Hind III or EcoR I and electrophoresed on a 1% agarose gel to identify clones. The identified clones were designated as "pET / α-1,3 / β-1,4-GT".

The fusion protein produced by the expression of the fusion gene was secreted in the medium by using the α-galactosyltransferase signal sequence, and α-1,3-galactosyltransferase and β-1 as shown in FIG. 1 . , 4-go were added to the two amino acid sequences (Leu-Glu) on the connection chamber of the galactosyl transferase, β-1,4- galactosyl transferase terminal has added a histidine tag (His ₆ -tag) sequence . The present inventors named the fusion protein prepared above as "α-1,3 / β-1,4 galactosyltransferase".

Example 2 Expression and Purification of α-1,3 / β-1,4-galactosyltransferase

The inventors of the present invention comprises a vector (pET / α-1,3 / β-1,4-GT) containing the fusion protein prepared in Example 1, which is an ompT deficient host cell E. coli BL21 (DE3) ( Novagen, Darmstadt, Germany). Overnight transformed Escherichia coli BL21 (DE3) was diluted 1/100 in 20 ml of LB medium containing kanamycin at a concentration of 50 μg / ml, shaken at 37 ° C. at 250 rpm until A ₆₀₀ was 0.7 and IPTG was added at 3 mM concentration. By inducing expression.

Specifically, after incubation at 37 ° C. for 4-5 hours after the addition of IPTG, the cells were recovered by centrifugation, suspended in 3 ml of 30 mM Tris-HCl, 30 mM NaCl solution (pH 7.5), and left on ice for 10 minutes to expand. I was. Centrifuge the expanded strain, discard the supernatant, suspend the strain in 1-2 ml of lysis buffer I (1 mM Tris-HCl (pH 7.5), 2% NP40), and grind four times for 4 seconds using an ultrasonic digester. The supernatant was recovered by centrifugation at 15,000 rpm. To the remaining insoluble protein, add 1-3 ml of lysis buffer II (8 M urea or 1 mM β-mercaptoethanol, 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA (pH 8.0)) After mixing well and crushed three times again for 10 seconds, and centrifuged at 15,000 rpm to collect the supernatant. The combined supernatants were placed in a permeable membrane and dialyzed in ₁ × PBS (6.7 mM K ₂ HPO ₄ , 150 mM NaCl, pH 7.4) in a cold chamber for 14-16 hours. Each aqueous and insoluble fraction was analyzed by 10% sodium dodecyl sulgate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis gel was stained with gel staining solution (0.025% Coomassie Brilliant Blue R, 250, 40% methanol, 7% acetic acid) for 1 hour, and then decolorized solution (30% methanol, 7% acetic acid, 63% H ₂ O). It bleached.

As a result, when the cell culture at 37 ℃ 60% of the fusion protein was expressed insoluble, only 40% was expressed as a water-soluble protein ( Fig. 2 ). In addition, when the expression was induced by IPTG, the main band was expressed in α-1,3 / β-1,4-galactosyltransferase at 85 kDa ( Fig . 3e).

Western blot analysis was performed using an anti-HisTag antibody with a histidine tag (His · Tag) portion of β-1,4-galactosyltransferase of the fusion protein, and α-1,3-galactosyltransferase Anti S. Tag antibody was used. The protein-transferred membrane was coated with 5% skim milk powder and washed three times with TTBS (20 mM Tris-HCl, pH 7.6), 150 mM NaCl, 0.05% tween 20) for 15 minutes. The anti-His-Tag antibody and the anti-S-Tag antibody obtained from the mice were mixed with TTBS solution at 5 µg / ml, and then bound to the transferred membrane for 1 hour at room temperature, and washed three times with TTBS solution for 15 minutes. To detect the first reacted anti-HisTag antigen, 0.06 U / ml of anti mouse IgG-POD obtained from sheep was mixed in a TTBS solution, bound to the transferred membrane for 1 hour at room temperature, and washed. The treated membrane was bound to a super signal west pico chemiluminescent substrate (PIERCE, U.S.A.) at room temperature for 5 minutes and then subjected to Kodak film (13 × 18 cm).

Each pET / β-1,4-GalT and fusion protein (pET / α-1,3-GalT / β-1,4-GalT) have His-Tag moiety, and 5 ml Ni-containing supernatant containing water-soluble protein. Loaded into NTA column. After washing the column with a dissolution buffer, the protein was washed with washing buffer (50 mM NaH ₂ PO ₄ , 20 mM imidazole, 300 mM NaCl, pH 8.0), and the beads were immediately used for the enzyme activity experiment.

As a result, α-1,3-galactosyltransferase was expressed at 42 kDa and β-1,4-galactosyltransferase was expressed at 50 kDa when expression was induced by IPTG. In addition, the major band of α-1,3 / β-1,4-galactosyltransferase was found to be expressed mainly at 85 kDa (b, d, f of Figure 3 ).

Example 3 Analysis of Enzyme Activity of α-1,3 / β-1,4-galactosyltransferase

The present inventors analyzed the enzymatic activity of α-1,3 / β-1,4-galactosyltransferase purified in Example 2. Galactosyltransferase enzyme activity analysis was performed by modifying the method reported by Do et al., 1995, J. Biological Chem. 270, 18447-18451.

Specifically, the enzymatic activity of the purified protein was determined in 0.1 M Na-cacodylic acid, pH 6.7, 10 mM MnCl ₂ , 10 mM ATP, N -acetylglucosamine 1 mM and already dried in Eppendorf tubes. Measurements were made in vitro by 50 μl reaction solution containing 1.6 pmol (1 × 10 ⁵ cpm) of [ ³ H] UDP-α-D-galactose (UDP-Gal). After 1 hour of reaction at 37 ° C., 1 ml of distilled water was added to terminate the reaction. The reaction mixture was loaded into a 1 ml Dowex, AG1-X8 pipette column previously equilibrated with 5% sodium borate. The column was washed five times with 1 ml of the same solution as above, followed by tritium-labeled galactose-β1-4 N -acetylglucosamine (Galβ1-4Glc N Ac), tritium-labeled galactose-α-1, in each fraction. 3-galactose-β1-4 N -acetylglucosamine (Galα1-3Galβ1-4Glc N Ac) was quantified with a liquid scintillation counter. To identify the reaction product disaccharides (Galβ1-4Glc N Ac) and trisaccharides (Galα1-3Galβ1-4Glc N Ac), the reaction mixture was previously equilibrated with 100 mM ammonium hydrogen carbonate (pH 7.7). It was loaded on 4 columns (Bio-Gel P-4 column; 1.5 × 100 cm). Tritium-labeled galactose inserted in disaccharides and trisaccharides was measured using a Liquidman scintillation counter LS6500.

As a result, as shown in FIG. 5 , the reaction product disaccharide (fraction 160-170) and trisaccharide (fraction 150-160) were separated from free galactose (fraction 190-200), and α-1-3 / β- prepared above. 1,4-galactosyltransferase has been shown to effectively form trisaccharides from monosaccharide N -acetylglucosamine.

As described above, the method for producing α1-3 / β-1,4-galactosyltransferase of the present invention can be mass-produced in the form of a water-soluble protein showing complete enzymatic activity using an ompT deficient host cell. Α-1-3 / β-1,4-galactosyltransferase prepared by galactose-β-1,4- N -acetylglucosamine and galactose-α-1,3 galactose-β-1,4- N -acetyl It can be usefully used to prepare a saccharide having a glucosamine structure.

1 is a schematic diagram showing a fusion protein of the present invention produced an α-1,3- galactosyl transferase and β-1,4- galactosyl transferase using the non-functional links.

2 α-1,3-galactosyltransferase (α-1,3-GalT), β-1,4-galactosyltransferase (β-1,4-GalT) and the fusion protein of the present invention in E. coli After expression, the aqueous supernatant and the insoluble precipitate were separated and analyzed by SDS-PAGE.

3 is α-1,3-galactosyltransferase (α-1,3-GalT), β-1,4-galactosyltransferase (β-1,4-GalT) and the fusion protein of the present invention in E. coli It is expressed by IPTG, purified by NiNTA-column and analyzed by SDS-PAGE.

(a), (c), (e): Coomassie Blue Dyeing,

(b), (d), (f): Western blot using anti histidine tagged antibody

-: Non - IPTG, +: expression in the presence of IPTG

4 is a graph showing the degree of enzymatic activity of beta mercaptoethanol of the fusion enzyme produced by the method of the present invention.

Precipitation 1: fusion protein dissolved in grinding buffer I (2% NP 40, 1 mM Tris-HCl),

Precipitation 2: A fusion protein which was dissolved in the ground buffer I again in the ground buffer II (2% NP 40, 1 mM Tris-HCl, 1 mM β-mercaptoethanol)

5 is a disaccharide galactose-β-1 synthesized by α-1,3-galactosyltransferase, β-1,4-galactosyltransferase produced by the method of the present invention and the fusion protein of the present invention. , 4- N -acetylglucosamine (Gal-β-1,4-GlcNAc) and trisaccharide galactose-α-1,3-galactose-β-1,4- N -acetylglucosamine (Gal-α-1,3- Gal-β-1,4-GlcNAc).

<110> Korea Research Institute of Bioscience and Biotechnology <120> Fusion protein producing galactose alpha-1,3-galactose-beta-1,4-N-acetyl glucosamine <130> 2p-08-03 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 1185 <212> DNA <213> Mus musculus <400> 1 atgatcacta tgcttcaaga tctccatgtc aacaagatct ccatgtcaag atccaagtca 60 gaaacaagtc ttccatcctc aagatctgga tcacaggaga aaataatgaa tgtcaaggga 120 aaagtaatcc tgttgatgct gattgtctca accgtggttg tcgtgttttg ggaatatgtc 180 aacagaattc cagaggttgg tgagaacaga tggcagaagg actggtggtt cccaagctgg 240 tttaaaaatg ggacccacag ttatcaagaa gacaacgtag aaggacggag agaaaagggt 300 agaaatggag atcgcattga agagcctcag ctatgggact ggttcaatcc aaagaaccgc 360 ccggatgttt tgacagtgac cccgtggaag gcgccgattg tgtgggaagg cacttatgac 420 acagctctgc tggaaaagta ctacgccaca cagaaactca ctgtggggct gacagtgttt 480 gctgtgggaa agtacattga gcattactta gaagactttc tggagtctgc tgacatgtac 540 ttcatggttg gccatcgggt catattttac gtcatgatag acgacacctc ccggatgcct 600 gtcgtgcacc tgaaccctct acattcctta caagtctttg agatcaggtc tgagaagagg 660 tggcaggata tcagcatgat gcgcatgaag accattgggg agcacatcct ggcccacatc 720 cagcacgagg tcgacttcct cttctgcatg gacgtggatc aagtctttca agacaacttc 780 ggggtggaaa ctctgggcca gctggtagca cagctccagg cctggtggta caaggccagt 840 cccgagaagt tcacctatga gaggcgggaa ctgtcggccg cgtacattcc attcggagag 900 ggggattttt actaccacgc ggccattttt ggaggaacgc ctactcacat tctcaacctc 960 accagggagt gctttaaggg gatcctccag gacaagaaac atgacataga agcccagtgg 1020 catgatgaga gccacctcaa caaatacttc cttttcaaca aacccactaa aatcctatct 1080 ccagagtatt gctgggacta tcagataggc ctgccttcag atattaaaag tgtcaaggta 1140 gcttggcaga caaaagagta taatttggtt agaaataatg tctga 1185 <210> 2 <211> 376 <212> PRT <213> Mus musculus <400> 2 Met Ile Thr Met Leu Gln Asp Leu His Val Asn Lys Ile Ser Met Ser 1 5 10 15 Arg Ser Lys Ser Glu Thr Ser Leu Pro Ser Ser Arg Ser Gly Ser Gln 20 25 30 Glu Lys Ile Met Asn Val Lys Gly Lys Val Ile Leu Leu Met Leu Ile 35 40 45 Val Ser Thr Val Val Val Val Phe Trp Glu Tyr Val Asn Arg Ile Pro 50 55 60 Glu Val Gly Glu Asn Arg Trp Gln Asp Asn Val Glu Gly Arg Arg Glu 65 70 75 80 Lys Gly Arg Asn Gly Asp Arg Ile Glu Glu Pro Gln Leu Trp Asp Trp 85 90 95 Phe Asn Pro Lys Asn Arg Pro Asp Val Leu Thr Val Thr Pro Trp Lys 100 105 110 Ala Pro Ile Val Trp Glu Gly Thr Tyr Asp Thr Ala Leu Leu Glu Lys 115 120 125 Tyr Tyr Ala Thr Gln Lys Leu Thr Val Gly Leu Thr Val Phe Ala Val 130 135 140 Gly Lys Tyr Ile Glu His Tyr Leu Glu Asp Phe Leu Glu Ser Ala Asp 145 150 155 160 Met Tyr Phe Met Val Gly His Arg Val Ile Phe Tyr Val Met Ile Asp 165 170 175 Asp Thr Ser Arg Met Pro Val Val His Leu Asn Pro Leu His Ser Leu 180 185 190 Gln Val Phe Glu Ile Arg Ser Glu Lys Arg Trp Gln Asp Ile Ser Met 195 200 205 Met Arg Met Lys Thr Ile Gly Glu His Ile Leu Ala His Ile Gln His 210 215 220 Glu Val Asp Phe Leu Phe Cys Met Asp Val Asp Gln Val Phe Gln Asp 225 230 235 240 Asn Phe Gly Val Glu Thr Leu Gly Gln Leu Val Ala Gln Leu Gln Ala 245 250 255 Trp Trp Tyr Lys Ala Ser Pro Glu Lys Phe Thr Tyr Glu Arg Arg Glu 260 265 270 Leu Ser Ala Ala Tyr Ile Pro Phe Gly Glu Gly Asp Phe Tyr Tyr His 275 280 285 Ala Ala Ile Phe Gly Gly Thr Pro Thr His Ile Leu Asn Leu Thr Arg 290 295 300 Glu Cys Phe Lys Gly Ile Leu Gln Asp Lys Lys His Asp Ile Glu Ala 305 310 315 320 Gln Trp His Asp Glu Ser His Leu Asn Lys Tyr Phe Leu Phe Asn Lys 325 330 335 Pro Thr Lys Ile Leu Ser Pro Glu Tyr Cys Trp Asp Tyr Gln Ile Gly 340 345 350 Leu Pro Ser Asp Ile Lys Ser Val Lys Val Ala Trp Gln Thr Lys Glu 355 360 365 Tyr Asn Leu Val Arg Asn Asn Val 370 375 <210> 3 <211> 1197 <212> DNA <213> Homo sapiens <400> 3 atgaggcttc gggagccgct cctgagcggc agcgccgcga tgccaggcgc gtccctacag 60 cgggcctgcc gcctgctcgt ggccgtctgc gctctgcacc ttggcgtcac cctcgtttac 120 tacctggctg gccgcgacct gagccgcctg ccccaactgg tcggagtctc cacaccgctg 180 cagggcggct cgaacagtgc cgccgccatc gggcagtcct ccggggagct ccggaccgga 240 ggggcccggc cgccgcctcc tctaggcgcc tcctcccagc cgcgcccggg tggcgactcc 300 agcccagtcg tggattctgg ccctggcccc gctagcaact tgacctcggt cccagtgccc 360 cacaccaccg cactgtcgct gcccgcctgc cctgaggagt ccccgctgct tgtgggcccc 420 atgctgattg agtttaacat gcctgtggac ctggagctcg tggcaaagca gaacccaaat 480 gtgaagatgg gcggccgcta tgcccccagg gactgcgtct ctcctcacaa ggtggccatc 540 atcattccat tccgcaaccg gcaggagcac ctcaagtact ggctatatta tttgcaccca 600 gtcctgcagc gccagcagct ggactatggc atctatgtta tcaaccaggc gggagacact 660 atattcaatc gtgctaagct cctcaatgtt ggctttcaag aagccttgaa ggactatgac 720 tacacctgct ttgtgtttag tgacgtggac ctcattccaa tgaatgacca taatgcgtac 780 aggtgttttt cacagccacg gcacatttcc gttgcaatgg ataagtttgg attcagccta 840 ccttatgttc agtattttgg aggtgtctct gctctaagta aacaacagtt tctaaccatc 900 aatggatttc ctaataatta ttggggctgg ggaggagaag atgatgacat ttttaacaga 960 ttagttttta gaggcatgtc tatatctcgc ccaaatgctg tggtcgggag gtgtcgcatg 1020 atccgccact caagagacaa gaaaaatgaa cccaatcctc agaggtttga ccgaattgca 1080 cacacaaagg agacaatgct ctctgatggt ttgaactcac tcacctacca ggtgctggat 1140 gtacagagat acccattgta tacccaaatc acagtggaca tcgggacacc gagctag 1197 <210> 4 <211> 398 <212> PRT <213> Homo sapiens <400> 4 Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ser Ala Ala Met Pro Gly 1 5 10 15 Ala Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala Val Cys Ala Leu 20 25 30 His Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu Ser 35 40 45 Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser 50 55 60 Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly 65 70 75 80 Gly Ala Arg Pro Pro Pro Pro Leu Gly Ala Ser Ser Gln Pro Arg Pro 85 90 95 Gly Gly Asp Ser Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser 100 105 110 Asn Leu Thr Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 115 120 125 Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile Glu 130 135 140 Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn 145 150 155 160 Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His 165 170 175 Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys 180 185 190 Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln Leu Asp 195 200 205 Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg 210 215 220 Ala Lys Leu Leu Asn Val Gly Phe Gln Glu Ala Leu Lys Asp Tyr Asp 225 230 235 240 Tyr Thr Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp 245 250 255 His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala 260 265 270 Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly 275 280 285 Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro 290 295 300 Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg 305 310 315 320 Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 325 330 335 Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro Asn 340 345 350 Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Leu Ser 355 360 365 Asp Gly Leu Asn Ser Leu Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr 370 375 380 Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Ser 385 390 395 <210> 5 <211> 2163 <212> DNA <213> Artificial Sequence <220> <223> cDNA for fusion protein (alpha 1,3 / beta 1,4-galactosyltransferase) <400> 5 atgaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagccc agatctgggt 60 accctggtgc cacgcggttc catggcgata attccagagg ttggtgagaa cagatggcag 120 aaggactggt ggttcccaag ctggtttaaa aatgggaccc acagttatca agaagacaac 180 gtagaaggac ggagagaaaa gggtagaaat ggagatcgca ttgaagagcc tcagctatgg 240 gactggttca atccaaagaa ccgcccggat gttttgacag tgaccccgtg gaaggcgccg 300 attgtgtggg aaggcactta tgacacagct ctgctggaaa agtactacgc cacacagaaa 360 ctcactgtgg ggctgacagt gtttgctgtg ggaaagtaca ttgagcatta cttagaagac 420 tttctggagt ctgctgacat gtacttcatg gttggccatc gggtcatatt ttacgtcatg 480 atagacgaca cctcccggat gcctgtcgtg cacctgaacc ctctacattc cttacaagtc 540 tttgagatca ggtctgagaa gaggtggcag gatatcagca tgatgcgcat gaagaccatt 600 ggggagcaca tcctggccca catccagcac gaggtcgact tcctcttctg catggacgtg 660 gatcaagtct ttcaagacaa cttcggggtg gaaactctgg gccagctggt agcacagctc 720 caggcctggt ggtacaaggc cagtcccgag aagttcacct atgagaggcg ggaactgtcg 780 gccgcgtaca ttccattcgg agagggggat ttttactacc acgcggccat ttttggagga 840 acgcctactc acattctcaa cctcaccagg gagtgcttta aggggatcct ccaggacaag 900 aaacatgaca tagaagccca gtggcatgat gagagccacc tcaacaaata cttccttttc 960 aacaaaccca ctaaaatcct atctccagag tattgctggg actatcagat aggcctgcct 1020 tcagatatta aaagtgtcaa ggtagcttgg cagacaaaag agtataattt ggttagaaat 1080 aatgtcctcg agcgcctgcc ccaactggtc ggagtctcca caccgctgca gggcggctcg 1140 aacagtgccg ccgccatcgg gcagtcctcc ggggagctcc ggaccggagg ggcccggccg 1200 ccgcctcctc taggcgcctc ctcccagccg cgcccgggtg gcgactccag cccagtcgtg 1260 gattctggcc ctggccccgc tagcaacttg acctcggtcc cagtgcccca caccaccgca 1320 ctgtcgctgc ccgcctgccc tgaggagtcc ccgctgcttg tgggccccat gctgattgag 1380 tttaacatgc ctgtggacct ggagctcgtg gcaaagcaga acccaaatgt gaagatgggc 1440 ggccgctatg cccccaggga ctgcgtctct cctcacaagg tggccatcat cattccattc 1500 cgcaaccggc aggagcacct caagtactgg ctatattatt tgcacccagt cctgcagcgc 1560 cagcagctgg actatggcat ctatgttatc aaccaggcgg gagacactat attcaatcgt 1620 gctaagctcc tcaatgttgg ctttcaagaa gccttgaagg actatgacta cacctgcttt 1680 gtgtttagtg acgtggacct cattccaatg aatgaccata atgcgtacag gtgtttttca 1740 cagccacggc acatttccgt tgcaatggat aagtttggat tcagcctacc ttatgttcag 1800 tattttggag gtgtctctgc tctaagtaaa caacagtttc taaccatcaa tggatttcct 1860 aataattatt ggggctgggg aggagaagat gatgacattt ttaacagatt agtttttaga 1920 ggcatgtcta tatctcgccc aaatgctgtg gtcgggaggt gtcgcatgat ccgccactca 1980 agagacaaga aaaatgaacc caatcctcag aggtttgacc gaattgcaca cacaaaggag 2040 acaatgctct ctgatggttt gaactcactc acctaccagg tgctggatgt acagagatac 2100 ccattgtata cccaaatcac agtggacatc gggacaccga gccatcatca tcatcatcat 2160 tag 2163 <210> 6 <211> 720 <212> PRT <213> Artificial Sequence <220> <223> fusion protein (alpha 1,3 / beta 1,4-galactosyltransferase) <400> 6 Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser 1 5 10 15 Pro Asp Leu Gly Thr Leu Val Pro Arg Gly Ser Met Ala Ile Ile Pro 20 25 30 Glu Val Gly Glu Asn Arg Trp Gln Lys Asp Trp Trp Phe Pro Ser Trp 35 40 45 Phe Lys Asn Gly Thr His Ser Tyr Gln Glu Asp Asn Val Glu Gly Arg 50 55 60 Arg Glu Lys Gly Arg Asn Gly Asp Arg Ile Glu Glu Pro Gln Leu Trp 65 70 75 80 Asp Trp Phe Asn Pro Lys Asn Arg Pro Asp Val Leu Thr Val Thr Pro 85 90 95 Trp Lys Ala Pro Ile Val Trp Glu Gly Thr Tyr Asp Thr Ala Leu Leu 100 105 110 Glu Lys Tyr Tyr Ala Thr Gln Lys Leu Thr Val Gly Leu Thr Val Phe 115 120 125 Ala Val Gly Lys Tyr Ile Glu His Tyr Leu Glu Asp Phe Leu Glu Ser 130 135 140 Ala Asp Met Tyr Phe Met Val Gly His Arg Val Ile Phe Tyr Val Met 145 150 155 160 Ile Asp Asp Thr Ser Arg Met Pro Val Val His Leu Asn Pro Leu His 165 170 175 Ser Leu Gln Val Phe Glu Ile Arg Ser Glu Lys Arg Trp Gln Asp Ile 180 185 190 Ser Met Met Arg Met Lys Thr Ile Gly Glu His Ile Leu Ala His Ile 195 200 205 Gln His Glu Val Asp Phe Leu Phe Cys Met Asp Val Asp Gln Val Phe 210 215 220 Gln Asp Asn Phe Gly Val Glu Thr Leu Gly Gln Leu Val Ala Gln Leu 225 230 235 240 Gln Ala Trp Trp Tyr Lys Ala Ser Pro Glu Lys Phe Thr Tyr Glu Arg 245 250 255 Arg Glu Leu Ser Ala Ala Tyr Ile Pro Phe Gly Glu Gly Asp Phe Tyr 260 265 270 Tyr His Ala Ala Ile Phe Gly Gly Thr Pro Thr His Ile Leu Asn Leu 275 280 285 Thr Arg Glu Cys Phe Lys Gly Ile Leu Gln Asp Lys Lys His Asp Ile 290 295 300 Glu Ala Gln Trp His Asp Glu Ser His Leu Asn Lys Tyr Phe Leu Phe 305 310 315 320 Asn Lys Pro Thr Lys Ile Leu Ser Pro Glu Tyr Cys Trp Asp Tyr Gln 325 330 335 Ile Gly Leu Pro Ser Asp Ile Lys Ser Val Lys Val Ala Trp Gln Thr 340 345 350 Lys Glu Tyr Asn Leu Val Arg Asn Asn Val Leu Glu Arg Leu Pro Gln 355 360 365 Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser Asn Ser Ala Ala 370 375 380 Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly Gly Ala Arg Pro 385 390 395 400 Pro Pro Pro Leu Gly Ala Ser Ser Gln Pro Arg Pro Gly Gly Asp Ser 405 410 415 Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser Asn Leu Thr Ser 420 425 430 Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro Ala Cys Pro Glu 435 440 445 Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile Glu Phe Asn Met Pro 450 455 460 Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn Val Lys Met Gly 465 470 475 480 Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His Lys Val Ala Ile 485 490 495 Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys Tyr Trp Leu Tyr 500 505 510 Tyr Leu His Pro Val Leu Gln Arg Gln Gln Leu Asp Tyr Gly Ile Tyr 515 520 525 Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg Ala Lys Leu Leu 530 535 540 Asn Val Gly Phe Gln Glu Ala Leu Lys Asp Tyr Asp Tyr Thr Cys Phe 545 550 555 560 Val Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp His Asn Ala Tyr 565 570 575 Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala Met Asp Lys Phe 580 585 590 Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly Val Ser Ala Leu 595 600 605 Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro Asn Asn Tyr Trp 610 615 620 Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg Leu Val Phe Arg 625 630 635 640 Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly Arg Cys Arg Met 645 650 655 Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro Asn Pro Gln Arg Phe 660 665 670 Asp Arg Ile Ala His Thr Lys Glu Thr Met Leu Ser Asp Gly Leu Asn 675 680 685 Ser Leu Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr Pro Leu Tyr Thr 690 695 700 Gln Ile Thr Val Asp Ile Gly Thr Pro Ser His His His His His His His 705 710 715 720 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer with NcoI <400> 7 gagaccatgg ggaattccag aggttggtg 29 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> reverse primer with XhoI <400> 8 gtgtctcgag gacattattt ctaaccaaat tatcc 35

Claims (10)

1,3- α-galactosyl transferase in the fusion protein and comprising a β-1,4- galactosyl transferase, α-1,3- galactosyl transferase is 63 to one of the SEQ ID NO: 2 And 414 amino acid sequence, and β-1,4-galactosyltransferase has a amino acid sequence of SEQ ID NO: 49 to 398. delete delete delete delete The fusion protein of claim 1, wherein the fusion protein has an amino acid sequence as set forth in SEQ ID NO: 6 . The gene encoding the fusion protein of claim 1. 8. The gene according to claim 7, wherein the gene has a nucleotide sequence set forth in SEQ ID NO: 5 . 1) preparing an expression vector comprising the gene of claim 7 and histidine tag; 2) transforming the expression vector into ompT deficient E. coli; 3) culturing the transformant and inducing expression of α-1,3 / β-1,4-galactosyltransferase; And 4) A method for producing α-1,3 / β-1,4-galactosyltransferase comprising purifying the α-1,3 / β-1,4-galactosyltransferase. The galactose -β-1,4- using the fusion protein of claim 1 N - acetylglucosamine (galactose-β-1,4- N -acetylglucosamine ) -α-1,3 galactose and galactose -β-1,4- N A method for producing a functional complex sugar having a acetylglucosamine (Galactose-α-1,3-galactose-β-1,4- N -acetylglucosamine) structure.