KR100495126B1 - Fusion protein containing a activity of N-acetylgalactosaminyltransferase, a gene encoding the fusion protein and a transformed cell line transfected by the gene - Google Patents

Abstract

The present invention provides a fusion protein having an N-acetylgalactosaminiltransferase activity in which α-lactalbumin and β1,4-galactosyltransferase-1 are fused to each other, a gene encoding the same, a transformed cell line into which the gene is introduced, and The present invention relates to a method for producing a recombinant protein having a LacdiNAc-R glycoside structure using the transformed cell line, wherein the fusion protein or transformed cell line having the N-acetylgalactosaminiltransferase activity of the present invention is LacdiNAc-R glycoside structure. It can be usefully used for the production of recombinant protein having a.

Description

Fusion protein containing a activity of N-acetylgalactosaminyltransferase, a gene encoding the fusion protein and a transformed cell line transfected with N-acetylgalactosminyltransferase activity by the gene}

The present invention provides a fusion protein having an N-acetylgalactosaminiltransferase activity in which α-lactalbumin and β1,4-galactosyltransferase-1 are fused to each other, a gene encoding the same, a transformed cell line into which the gene is introduced, and The present invention relates to a method for producing a recombinant protein having a LacdiNAc-R sugar structure using the transformed cell line.

N-glycosylation and O-glycosylation glycostructures present in most glycoproteins are characterized by galactose-β1,4-GlcNAc (galactose-β1,4-GlcNAc; N- It exists as acetyllactosamine (LacNAc), and this structure serves as a mediator for making functional terminal glycosides such as sialyl-lewis, which exhibit specific physiological functions. Recently, however, a new structure has been found that is very similar to the above structure but plays a completely different role, which is the LacdiNAc-R complex sugar structure. LacdiNAc-R complex sugar structure is GalNAc- with N-acetylgalactosamine (GalNAc), which is N-acetylated to galactose instead of galactose, with β1,4-linkage to GlcNAc. β1,4-GlcNAc (N, N'-diacetyllactosdiamine, lacdiNAc) structure. The structure may optionally include a fucose connected to GlcNAc by α1,3-linking to form LacdiNAc ^x , and sulfate to GalNAc to form sulfated LacdiNAc-R sugar. The structure is made. The main human glycoproteins having the former structure (lacdiNAc-R) include luteinizing hormone, follicle stimulating hormone (FSH), thyrotrophins, and the latter structure (LacdiNAc ^x ). It is present in protein C, which has anticoagulant effect, and glycoderin, which has immunosuppressive and contraceptive effects. However, the production of such a glycoprotein human drug is very difficult in the absence of an appropriate cell line in the state in which the enzyme for producing LacdiNAc-R glycostructure is not known yet. Therefore, the enzyme for producing the LacdiNAc-R glycostructure is developed. There is an urgent need to develop transgenic cell lines that can be expressed continuously.

Accordingly, the present inventors fused a-lactalbumin and β1,4-galactosyltransferase-1 to a fusion protein having N-acetylgalactoaminyltransferase activity, a gene thereof, and a transformed cell line expressing the fusion protein. The present invention was completed by preparing a protein having a LacdiNAc-R glycoside structure using the transformed cell line.

The present invention provides a fusion protein having N-acetylgalactoaminyltransferase activity, a gene encoding the fusion protein, a transformed cell line into which the gene is introduced, and a recombinant having a LacdiNAc-R glycoside structure using the transformed cell line. It is to provide a method for producing a protein.

In order to achieve the above object, the present invention provides a fusion protein having α-lactalbumin and β1,4-galactosyltransferase-1 fused with N-acetylgalactosaminiltransferase activity.

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

The present invention also provides a transformed cell line in which the gene is introduced to biosynthesize LacdiNAc-R glycostructure.

In addition, the present invention provides a method for producing a recombinant protein having a LacdiNAc-R sugar structure using the transformed cell line.

Hereinafter, the present invention will be described in detail.

The present invention fuses α-lactalbumin with β1,4-galactosyltransferase-1 to provide a fusion protein having N-acetylgalactosaminiltransferase (lacdiNAc-T) activity.

The fusion protein linked to α-lactalbumin and β1,4-galactosyltransferase-1 of the present invention is N-acetylgalactosamineltransferase which is not present in α-lactalbumin and β1,4-galactosyltransferase-1. It is possible to produce a protein having an activity and having a LacdiNAc-R (GalNAc-β1,4-GlcNAc-R) sugar structure by the activity. In addition, the fusion proteins of the present invention have lactosyltransferase (lac-T) activity and galactosyltransferase (gal-T) activity as well as N-acetylgalactosaminiltransferase activity. In the above, N-acetylgalactosminyltransferase activity and lactosyltransferase activity are new activities that are not present in existing proteins.

In the present invention, the α-lactalbumin is α-lactalbumin having an amino acid sequence represented by SEQ ID NO: 2 (GeneBank No; J05147), and the β1,4-galactosyltransferase-1 is SEQ ID NO: 4 (GeneBank No X55415), the amino acid sequence described above, and preferably include a cytoplasmic site of β1,4-galactosyltransferase-1 and a site from which the transmembrane site has been removed, and from SEQ ID NO: 49 to 398 More preferably it comprises an amino acid sequence. Further, the fusion protein of the present invention most preferably comprises the sequence set forth in SEQ ID NO: 7 excluding the signal sequence of α-lactalbumin. However, the fusion protein of the present invention is not limited to the above sequence, and LacdiNAc has N-acetylgalactosaminiltransferase activity in a fusion protein in which α-lactalbumin and β1,4-galactosyltransferase-1 are fused. -R (GalNAc-β1,4-GlcNAc-R) Includes all fusion proteins that biosynthesize the sugar structure.

In the exemplary embodiment of the present invention, a linkage between bovine α-lactalbumin and β1,4-galactosyltransferase-1 is a non-functional linker containing a restriction enzyme recognition sequence and a two amino acid sequence (Leu-Glu). was added, the end of β1,4- galactosyl transferase -1 in order to facilitate the isolation and purification of the fusion protein, by adding a histidine tag (His ₆ -tag) comprising the amino acid sequence described in SEQ ID NO: 6 Fusion proteins were prepared (see FIG. 1 ).

The present invention provides a gene encoding a fusion protein having the above N-acetylgalactosamineyltransferase (lacdiNAc-T) activity.

N-acetylgalactosaminyltransferase (lacdiNAc-T) activity by linking cDNA of α-lactalbumin described in SEQ ID NO: 1 with β1,4-galactosyltransferase-1 described in SEQ ID NO: 3 in the present invention It provides a gene expressing a fusion protein having a.

In the present invention, the gene is preferably a gene which connect the cDNA with β1,4- galactosyl cytoplasmic region and the membrane penetration region is removed cDNA of acyltransferase-1 of α- lactalbumin, and described in SEQ ID NO: 5 It is preferable that it is a gene having a nucleotide sequence. However, the genes of the present invention are not limited to the above sequences, but include genes encoding all fusion proteins having N-acetylgalactosaminiltransferase activity.

In an embodiment of the present invention, the cDNA of α-lactalbumin and the cDNA from which the cytoplasmic site and the transmembrane site of the β1,4-galactosyltransferase-1 are removed are connected, and the α-lactalbumin and β1,4-galacto A non-functional linker was added to the linkage of the siltransferase gene, and a base sequence corresponding to the two amino acid sequences (Leu-Glu) was added, and a histidine tag (His ₆ -tag) was attached to the end of β1,4-galactosyltransferase. A fusion gene was prepared to which the corresponding base sequence was added. The fusion gene was named "α-LAGT".

The present invention provides a transformed cell line capable of biosynthesizing LacdiNAc-R sugar structure by introducing the gene encoding the fusion protein.

In the embodiment of the present invention, the α-LAGT fusion gene was liposomal-mediated gene transfection into the Lec8 cell line (ATCC) to obtain a single cell line capable of continuously expressing the fusion protein. The Lec8 cell line is a cell line deficient in galactosylation of the glycoprotein of the cell line itself. Thus, the Lec8 cell line can provide an in vivo substrate very appropriately for the lacdiNAc-T activity of the novel fusion protein of the present invention. There is an advantage.

The present inventors named the transformed cell line into which the α-LAGT fusion gene was introduced, "Lec8 / LG60", and deposited it on April 12, 2002 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10223BP).

The present invention provides a method for producing a recombinant protein having a LacdiNAc-R glycostructure using the transformed cell line.

Method for producing a protein having a LacdiNAc-R glycostructure of the present invention

1) introducing the gene of interest into the transformed cell line expressing the fusion protein of the present invention;

2) culturing the transformed cell line to mass express a protein encoding the gene of interest; And

3) separating and purifying the protein of interest from the culture solution.

In the embodiment of the present invention, the histidine tag (His ₆ ) was added to the COOH-terminus for pure separation of the fusion protein so that the expressed protein could be separated using a Ni-NTA-agarose affinity column. The expression level of the fusion protein was analyzed by Western blotting using an antibody that specifically reacts with α-lactalbumin. The transformed cell line was found to express a 62 kDa fusion protein (see FIG. 2 ). In addition, the activity of the fusion protein expressed in the transformed cell line, as well as galactosyltransferase (gal-T) activity, N-acetylgalactosaminiltransferase (LacdiNAc-T) activity and lactosyltransferase It was shown to have (lac-T) activity simultaneously (see FIG. 3 ). The present invention is the first time that a fusion gene is formed by combining the two genes, and a protein showing such enzymatic activity as a single polypeptide chain has not been reported yet. In addition, the transformed cell line expressing the fusion protein of the present invention produced a protein actually having a LacdiNAc-R glycostructure in vivo (see FIG. 4 ).

The fusion protein of the present invention makes LacdiNAc-R glycoside structure, is a new protein expressed in the fusion gene, and can be usefully used for the production of various recombinant proteins having LacdiNAc-R glycoside structure.

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 Preparation of α-LAGT Fusion Gene Linking c-DNA of α-Lactalbumin and cDNA of β1,4-galactosyltransferase

In order to prepare a fusion protein having N-acetylgalactosminyltransferase activity, the present inventors use the entire cDNA gene ( SEQ ID NO: 1 ; GeneBank Accession No .: J05147) and human β1, including a signal sequence of bovine α-lactalbumin. Fusion protein was prepared using 4-galactosyltransferase whole cDNA gene ( SEQ ID NO: 3 ; GeneBank Accession #: X55415).

Specifically, a 48 amino acid sequence which is a NH ₂ -terminal part corresponding to the cytoplasmic site and transmembrane site of the entire cDNA gene including the signal sequence of bovine α-lactalbumin and the entire cDNA gene of human β1,4-galactosyltransferase A fusion gene was prepared by fusion of the β1,4-galactosyltransferase (Arg ⁴⁹ to Ser ³⁹⁸ ) gene in a soluble form with 1: 1 removed.

The fusion protein produced by the expression of the fusion gene was secreted into the medium by using the α-lactalbumin signal sequence, and as shown in FIG. 1 , the linkage region of α-lactalbumin and β1,4-galactosyltransferase gene. Two amino acid sequences (Leu-Glu) were added to the non-functional linker, and histidine tag (His _6- tag) sequence was added to the β1,4-galactosyltransferase ends. We named the fusion gene "α-LAGT" ( Figure 1 ).

Example 2 Preparation of Transgenic Cell Line Expressing Fusion Protein

The present inventors prepared a transformed cell line capable of continuously expressing the fusion gene prepared in Example 1.

Specifically, the α-LAGT fusion gene prepared in Example 1 was obtained by liposomal-mediated gene transfection on the Lec8 cell line (ATCC) to obtain single cell lines LG10, LG35, and LG60 that are continuously expressed. The Lec8 cell line was a Lec8-T single cell line expressing the SV40 large-T antigen for high expression of recombinant protein, and by continuously expressing the α-LAGT fusion gene, the biosynthesis of LacdiNAc-R glycostructure in vivo I could do it.

Example 3 Analysis of Expression of Fusion Proteins of Transgenic Cell Lines

The inventors performed a Western blot to determine the expression pattern of the fusion protein in the transformed cell line prepared in Example 2. For pure separation of the fusion protein, histidine-tag (His ₆ ) was added to the COOH-terminus so that the expressed protein could be separated using a Ni-NTA-agarose affinity column. The expression level of the fusion protein was analyzed by Western blot using an antibody that specifically reacts with α-lactalbumin.

Specifically, the sustained-expressing cell line and the control cell line were washed with 1XPBS buffer (6.7 mM KH ₂ PO ₄ , 150 mM NaCl, pH 7.4) and then dissolved in PBS buffer containing 0.1% NP-40 to dissolve the whole cell protein in SDS-. The separation was carried out by PAGE electrophoresis. After transferring the isolated protein to the nitrocellulose membrane, rabbit α-lactalbumin specific antibody (1,500 dilution, primary Ab) and anti-rabbit IgG-peroxidase (IgG-peroxidase; 1; 4000, secondary Ab) ECL (Enhanced chemiluminescence, Amersham) was performed using.

As a result, LG10, LG35 and LG60 transformed cell lines expressed a 62 kDa fusion protein, of which LG60 expressed the most fusion protein ( FIG. 2 ).

Example 4 Determination of Enzyme Activity of a Fusion Protein

The present inventors, in order to determine the enzymatic activity of the fusion protein expressed in the transformed cell line prepared in Example 2, after separating the fusion protein of the present invention galactosyltransferase activity, lactosyltransferase activity and N-acetylgalactosaminiltransferase activity was measured.

Specifically, the fusion protein secreted in the medium was purely separated using Ni-NTA-agarose, washed with 1XPBS buffer, and then Ni-bead was used for direct enzymatic activity measurement. The reaction conditions were 100 mM Na-cacodylate buffer containing 20 mM MnCl ₂ using 20 mM GlcNAc and 0.1 μCi [6- ³ H] UDP-galactose in a total 50 μl reaction volume. pH 7.0) was reacted at 37 ° C. for 2 hours, and then the reaction product was passed through a Dowex 1 × 8 (Cl ⁻ form) column and Sepharose 4B column to measure the cpm value of the reaction product. In addition, lactosyltransferase enzyme activity was measured using [6- ³ H] UDP-galactose and 20 mM glucose as a substrate under the same conditions as mentioned above, and N-acetylgalactosaminiltransferase enzyme activity was measured by [6 ³ H] UDP-GalNAc and GlcNAc were measured using donor and acceptor substrates, respectively.

As a result, the fusion protein of the present invention is shown to have not only galactosyltransferase activity but also N-acetylgalactosaminiltransferase activity and lactosyltransferase activity ( FIG. 3 ).

Example 5 LacdiNAc-R Glycostructure Formation Analysis of Fusion Proteins

In order to investigate whether the fusion protein of the present invention actually produces the LacdiNAc-R glycostructure, we analyzed the activity of the fusion protein using LacdiNAc-R saccharide-specific monoclonal antibody.

Specifically, LG60, LG35 and LG10 single cell lines were cultured in the presence of G418 (400 μg / ml) and blasticidine (10 μg / ml), and then the cell line and the control cell line (Lec8 mock, only vector) Transformed cell lines) were washed with 1XPBS buffer (6.7 mM KH ₂ PO ₄ , 150 mM NaCl, pH 7.4) and sonicated in 20 mM Na-cacodylate buffer (pH 7.0) containing 1 mM CaCl ₂ . After sonication, the nuclei were removed, ultracentrifugation was performed at 100,000Xg for 1 hour, and then microsomes were separated to perform MDS-PAGE electrophoresis on membrane glycoproteins. This was again transferred to the nitrocellulose membrane, followed by ECL (Enhanced) using LacdiNAc-R specific monoclonal antibody (1: 1000 dilution, primary Ab) and anti-mouse IgM-peroxidase (1; 4000, secondary Ab). chemiluminescence) was performed (photosensitive with Kodak BioMax MR1 film).

As a result, the control cell line did not show a single antibody reaction with the LacdiNAc-R glycostructure, but the transformed cell line of the present invention showed a strong reaction of the single antibody with the LacdiNAc-R glycostructure. 4 ). From the above results, the transformed cell line expressing the fusion protein of the present invention has N-acetylgalactosaminiltransferase activity in vivo and simultaneously produces a protein having LacdiNAc-R glycoside structure in the cell. I could see that.

As described above, the fusion protein of the α-lactalbumin and β1,4-galactosyltransferase-1 of the present invention is not only lactosyltransferase activity but also lactosyltransferase activity and LacdiNAc-R glycoside structure. Since it also has N-acetylgalactosamineyltransferase activity to form a, it can be very useful for the production of a recombinant protein having a LacdiNAc-R glycostructure and a recombinant human pharmaceutical protein having the LacdiNAc-R glycostructure.

1 is a schematic diagram showing the structure of β1,4-galactosyltransferase-1 and the structure of the α-LAGT fusion gene of the present invention,

Gal-T-1: whole gene of β1,4-galactosyltransferase-1,

s-Gal-T-1: partial gene of secreted type β1,4-galactosyltransferase-1,

cy: cytoplasmic site, m: transmembrane site, stem: stem site,

c: active part, s: signal sequence, ↓: signal sequence removal part,

Figure 2 is a photograph of the α-LAGT fusion gene introduced into the Lec8-T cell line, and then analyzed by Western blot of the fusion protein expressed from the transformed cell line,

Figure 3 is a graph measuring galactosyltransferase (gal-T) activity, lactosyltransferase (lac-T) activity and N-acetylgalactosaminiltransferase (lacdiNAc-T) activity of the fusion protein of the present invention ego,

4 is a photograph of Western blot using LacdiNAc-R specific monoclonal antibody to see if the fusion protein of the present invention makes LacdiNAc-R structure.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Fusion protein containing a activity of N-acetylgalactosaminyltransferase, a gene encoding the fusion protein and a transformed cell line transfected by the gene <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 724 <212> DNA <213> Bos taurus <400> 1 atttcagaat cttggggggt aaccaaaatg atgtcctttg tctctctgct cctggtaggc 60 atcctattcc atgccaccca ggctgaacag ttaacaaaat gtgaggtgtt ccgggagctg 120 aaagacttga agggctacgg aggtgtcagt ttgcctgaat gggtctgtac cacgtttcat 180 accagtggtt atgacacaca agccatagta caaaacaatg acagcacaga atatggactc 240 ttccagataa ataataaaat ttggtgcaaa gacgaccaga accctcactc aagcaacatc 300 tgtaacatct cctgtgacaa gttcctggat gatgatctta ctgatgacat tatgtgtgtc 360 aagaagattc tggataaagt aggaattaac tactggttgg cccataaagc actctgttct 420 gagaagctgg atcagtggct ctgtgagaag ttgtgaacac ctgctgtctt tgctgcttct 480 gtcctctttc tgttcctgga actcctctgc cccgtggcta cctcgttttg cttctttgta 540 cccccttgaa gctaactcgt ctctgagccc tgggccctgt agtgacaatg gacatgtaag 600 gactaatctc caggggtgca tgaatggcgc tctggacttt tgacccttss tcgatgtccc 660 tgatggcgct tttaatgcaa cagtacatat tccacttttg tcccgaataa aaagcctgat 720 tttg 724 <210> 2 <211> 142 <212> PRT <213> Bos taurus <220> <221> SIGNAL (222) (1) .. (19) <223> signal sequence <400> 2 Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala 1 5 10 15 Thr Gln Ala Glu Gln Leu Thr Lys Cys Glu Val Phe Arg Glu Leu Lys 20 25 30 Asp Leu Lys Gly Tyr Gly Gly Val Ser Leu Pro Glu Trp Val Cys Thr 35 40 45 Thr Phe His Thr Ser Gly Tyr Asp Thr Gln Ala Ile Val Gln Asn Asn 50 55 60 Asp Ser Thr Glu Tyr Gly Leu Phe Gln Ile Asn Asn Lys Ile Trp Cys 65 70 75 80 Lys Asp Asp Gln Asn Pro His Ser Ser Asn Ile Cys Asn Ile Ser Cys 85 90 95 Asp Lys Phe Leu Asp Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys 100 105 110 Lys Ile Leu Asp Lys Val Gly Ile Asn Tyr Trp Leu Ala His Lys Ala 115 120 125 Leu Cys Ser Glu Lys Leu Asp Gln Trp Leu Cys Glu Lys Leu 130 135 140 <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> 1500 <212> DNA <213> Artificial Sequence <220> <223> alpha-LAGT <400> 5 atgatgtcct ttgtctctct gctcctggta ggcatcctat tccatgccac ccaggctgaa 60 cagttaacaa aatgtgaggt gttccgggag ctgaaagact tgaagggcta cggaggtgtc 120 agtttgcctg aatgggtctg taccacgttt cataccagtg gttatgacac acaagccata 180 gtacaaaaca atgacagcac agaatatgga ctcttccaga taaataataa aatttggtgc 240 aaagacgacc agaaccctca ctcaagcaac atctgtaaca tctcctgtga caagttcctg 300 gatgatgatc ttactgatga cattatgtgt gtcaagaaga ttctggataa agtaggaatt 360 aactactggt tggcccataa agcactctgt tctgagaagc tggatcagtg gctctgtgag 420 aagttgctcg agcgcctgcc ccaactggtc ggagtctcca caccgctgca gggcggctcg 480 aacagtgccg ccgccatcgg gcagtcctcc ggggagctcc ggaccggagg ggcccggccg 540 ccgcctcctc taggcgcctc ctcccagccg cgcccgggtg gcgactccag cccagtcgtg 600 gattctggcc ctggccccgc tagcaacttg acctcggtcc cagtgcccca caccaccgca 660 ctgtcgctgc ccgcctgccc tgaggagtcc ccgctgcttg tgggccccat gctgattgag 720 tttaacatgc ctgtggacct ggagctcgtg gcaaagcaga acccaaatgt gaagatgggc 780 ggccgctatg cccccaggga ctgcgtctct cctcacaagg tggccatcat cattccattc 840 cgcaaccggc aggagcacct caagtactgg ctatattatt tgcacccagt cctgcagcgc 900 cagcagctgg actatggcat ctatgttatc aaccaggcgg gagacactat attcaatcgt 960 gctaagctcc tcaatgttgg ctttcaagaa gccttgaagg actatgacta cacctgcttt 1020 gtgtttagtg acgtggacct cattccaatg aatgaccata atgcgtacag gtgtttttca 1080 cagccacggc acatttccgt tgcaatggat aagtttggat tcagcctacc ttatgttcag 1140 tattttggag gtgtctctgc tctaagtaaa caacagtttc taaccatcaa tggatttcct 1200 aataattatt ggggctgggg aggagaagat gatgacattt ttaacagatt agtttttaga 1260 ggcatgtcta tatctcgccc aaatgctgtg gtcgggaggt gtcgcatgat ccgccactca 1320 agagacaaga aaaatgaacc caatcctcag aggtttgacc gaattgcaca cacaaaggag 1380 acaatgctct ctgatggttt gaactcactc acctaccagg tgctggatgt acagagatac 1440 ccattgtata cccaaatcac agtggacatc gggacaccga gccatcatca tcatcatcat 1500 1500 <210> 6 <211> 500 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <220> <221> SIGNAL (222) (1) .. (19) <223> signal sequence <220> <221> DOMAIN (222) (20) .. (142) <223> alpha-lactalbumin <220> <221> DOMAIN 222 (145) .. (494) <223> beta-1,4-galactosyltransfease-1 <220> <221> DOMAIN (222) (495) .. (500) <223> histidine tag <220> <221> SITE <222> (143) .. (144) <223> non-functional linker <400> 6 Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala 1 5 10 15 Thr Gln Ala Glu Gln Leu Thr Lys Cys Glu Val Phe Arg Glu Leu Lys 20 25 30 Asp Leu Lys Gly Tyr Gly Gly Val Ser Leu Pro Glu Trp Val Cys Thr 35 40 45 Thr Phe His Thr Ser Gly Tyr Asp Thr Gln Ala Ile Val Gln Asn Asn 50 55 60 Asp Ser Thr Glu Tyr Gly Leu Phe Gln Ile Asn Asn Lys Ile Trp Cys 65 70 75 80 Lys Asp Asp Gln Asn Pro His Ser Ser Asn Ile Cys Asn Ile Ser Cys 85 90 95 Asp Lys Phe Leu Asp Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys 100 105 110 Lys Ile Leu Asp Lys Val Gly Ile Asn Tyr Trp Leu Ala His Lys Ala 115 120 125 Leu Cys Ser Glu Lys Leu Asp Gln Trp Leu Cys Glu Lys Leu Leu Glu 130 135 140 Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser 145 150 155 160 Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly 165 170 175 Gly Ala Arg Pro Pro Pro Pro Leu Gly Ala Ser Ser Gln Pro Arg Pro 180 185 190 Gly Gly Asp Ser Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser 195 200 205 Asn Leu Thr Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro 210 215 220 Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile Glu 225 230 235 240 Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn 245 250 255 Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His 260 265 270 Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys 275 280 285 Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln Leu Asp 290 295 300 Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg 305 310 315 320 Ala Lys Leu Leu Asn Val Gly Phe Gln Glu Ala Leu Lys Asp Tyr Asp 325 330 335 Tyr Thr Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp 340 345 350 His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala 355 360 365 Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly 370 375 380 Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro 385 390 395 400 Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg 405 410 415 Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly 420 425 430 Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro Asn 435 440 445 Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Leu Ser 450 455 460 Asp Gly Leu Asn Ser Leu Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr 465 470 475 480 Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Ser His His 485 490 495 His His His His 500 <210> 7 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 7 Glu Gln Leu Thr Lys Cys Glu Val Phe Arg Glu Leu Lys Asp Leu Lys 1 5 10 15 Gly Tyr Gly Gly Val Ser Leu Pro Glu Trp Val Cys Thr Thr Phe His 20 25 30 Thr Ser Gly Tyr Asp Thr Gln Ala Ile Val Gln Asn Asn Asp Ser Thr 35 40 45 Glu Tyr Gly Leu Phe Gln Ile Asn Asn Lys Ile Trp Cys Lys Asp Asp 50 55 60 Gln Asn Pro His Ser Ser Asn Ile Cys Asn Ile Ser Cys Asp Lys Phe 65 70 75 80 Leu Asp Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys Lys Ile Leu 85 90 95 Asp Lys Val Gly Ile Asn Tyr Trp Leu Ala His Lys Ala Leu Cys Ser 100 105 110 Glu Lys Leu Asp Gln Trp Leu Cys Glu Lys Leu Leu Glu Arg Leu Pro 115 120 125 Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser Asn Ser Ala 130 135 140 Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly Gly Ala Arg 145 150 155 160 Pro Pro Pro Pro Leu Gly Ala Ser Ser Gln Pro Arg Pro Gly Gly Asp 165 170 175 Ser Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser Asn Leu Thr 180 185 190 Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro Ala Cys Pro 195 200 205 Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile Glu Phe Asn Met 210 215 220 Pro Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn Val Lys Met 225 230 235 240 Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His Lys Val Ala 245 250 255 Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys Tyr Trp Leu 260 265 270 Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln Leu Asp Tyr Gly Ile 275 280 285 Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg Ala Lys Leu 290 295 300 Leu Asn Val Gly Phe Gln Glu Ala Leu Lys Asp Tyr Asp Tyr Thr Cys 305 310 315 320 Phe Val Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp His Asn Ala 325 330 335 Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala Met Asp Lys 340 345 350 Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly Val Ser Ala 355 360 365 Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro Asn Asn Tyr 370 375 380 Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg Leu Val Phe 385 390 395 400 Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly Arg Cys Arg 405 410 415 Met Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro Asn Pro Gln Arg 420 425 430 Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Leu Ser Asp Gly Leu 435 440 445 Asn Ser Leu Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr Pro Leu Tyr 450 455 460 Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Ser 465 470 475

Claims (13)

A fusion protein set forth in SEQ ID NO: 6 or 7, wherein α-lactalbumin and β1,4-galactosyltransferase-1 are fused to have N-acetylgalactosamineltransferase (lacdiNAc-T) activity. The fusion protein of claim 1, wherein the fusion protein further has lactosyltransferase activity and galactosyltransferase activity. delete delete delete delete delete Gene encoding the fusion protein of claim 1. 9. The gene of claim 8, wherein the gene is set forth in SEQ ID NO: 5 . A transgenic cell line into which the gene of claim 8 is introduced. The method of claim 10, wherein the transformed cell line is a transformed cell line (accession number: KCTC 10223BP) characterized in that the gene of claim 9 is introduced into a single animal cell host. 1) introducing a gene encoding a recombinant protein into the transformed cell line of claim 10; 2) culturing the transformed cell line to mass express the recombinant protein; And 3) A method for producing a recombinant protein having a LacdiNAc-R (GalNAc-β1,4-GlcNAc-R) sugar structure, comprising separating and purifying the recombinant protein from the culture medium. The method according to claim 12, wherein the transformed cell line is the transformed cell line of claim 11.