BACKGROUND Most glycoproteins comprise N-glycosylation and 0-glycosylation structures in the form of galactose- 1,4-GlcNAc (N-acetyllactosamine, LacNAc) and the structure serves as a mediator for producing functional glycan epitope having specific physiological functions such as sialyl-Lewis, etc. Recently, a new structure, which is very similar to the above structure but serves totally different functions, has been disclosed. That is LacdiNAc-R (GalNAc-1, 4-GlcNAc-R) glycoprotein

structure. LacdiNAc-R glycoprotein complex has GalNAc- P 1, 4-GlcNAc (N, N'-diacetyllactosdiamine, lacdiNAc) structure in which N-acetylgalactosamine (GalNAc) is attached to GlcNAc by p 1, 4-linkage. Depending on circumstances, fucose is attached to GlcNAc by a 1,3- linkage, making LacdiNAcX or sulfated LacdiNAc-R glycoprotein) structure happen to be formed by the sulfation of GalNAc. Major human glycoproteins comprising the former structure (lacdiNAc-R or sulfated lacdiNAc-R) are exemplified by lutenizing hormone, FSH (follicle stimulating hormone), thyrotrophins, etc., and those comprising the latter structure (LacdiNAcX) are exemplified by protein C having anti-coagulation activity, glycodelin suppressing immune response and having contraceptive activity, etc. N- acetylgalactosaminyltransferase has been known as an enzyme producing LacdiNAc-R structure, yet a method for mass-production of the enzyme has not been established.

Moreover, host cells that can express the mentioned enzyme massively have not been found so far. Therefore, it is no wonder that glycoproteins comprising LacdiNAc- R structure have not been successfully used for producing medical supplies.

Thus, the present inventors prepared a fusion protein by combining a-lactalbumin with P 1,4-

galactosyltransferase-1, and transfected host cells with a gene encoding the fusion protein for the preparation of a transformed cell line. The present inventors completed the present invention by confirming that the transformed cells produced proteins comprising LacdiNAc-R glycoprotein structure.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention provides a fusion protein prepared by combining a-lactalbumin with ß 1,4- galactosyltransferase-1 and having the activities of N- acetylgalactosaminyltransferase, lactosyltransferas and glucosyltransferase.

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

This invention also provides transformed cells transfected by the above gene.

In addition, this invention provides a preparation method for producing target protein comprising LacdiNAc-R glycoprotein structure by using the above transformed cells.

Further features of the present invention will appear hereinafter.

The present invention provides a fusion protein

prepared by combining a-lactalbumin with P 1,4- galactosyltransferase-1 and having the activities of N- acetylgalactosaminyltransferase, lactosyltransferas and glucosyltransferase.

It is preferable for the region of the above a- lactalbumin to be the amino acid sequence represented by SEQ. ID. No 2 and for the region of the above P 1,4- galactosyltransferase-1 to be the amino acid sequence represented by SEQ. ID. No 4.

It is more preferable for the region of the above j3 1, 4-galactosyltransferase-1 to be the amino acid sequence represented by SEQ. ID. No 4 especially wherein cytosolic tail and transmembrane domain (lut- 48th amino acid region) are eliminated. Proteins can be secreted in water-soluble state when cytosolic tail and transmembrane domain are removed.

It is also preferable for the fusion protein of the present invention to contain a non-functional linker comprising restriction enzyme recognizing sequence additionally in order to combine amino acid sequences of a-lactalbumin with ß 1, 4- galactosyltransferase-1.

Therefore, it is preferable that the fusion protein of the present invention comprises a- lactalbumin sequence, a non-functional linker and an amino acid sequence represented by SEQ. ID. No 6

containing P 1, 4-galactosyltransferase-1 wherein cytosolic tail and transmembrane domain are eliminated.

When the fusion protein of the present invention is inserted in cells, signal sequence (from #1-to #19) at a-lactalbumin region represented by SEQ. ID.

No 2 is automatically removed, leading to the secretion of the fusion protein. Therefore, it is preferable that the fusion protein, as a final product secreted in cells, comprises a-lactalbumin sequence without signal sequence, a non-functional linker and an amino acid sequence represented by SEQ. ID. No 6 containing p 1,4- galactosyltransferase-1 wherein cytosolic tail and transmembrane domain are eliminated. However, the fusion protein of the present invention is not limited to the above mentioned sequences, yet can include every fusion protein that has the activity of N- acetylgalactosaminyltransferase and is able to synthesize LacdiNAc-R (GalNAc-P 1,4-GlcNAc-R) glycoprotein structure.

In the preferred embodiments of the present invention, the present inventors prepared a fusion protein by combining bovine a-lactalbumin amino acid sequence represented by SEQ. ID. No 2, a non-functional linker amino acid sequence (Leu-Glu) containing restriction enzyme recognition sequence, P 1,4- galactosyltransferase-1 amino acid sequence represented

by SEQ. ID. No 4 (from 49th to 398th amino acid) wherein cytosol and transmembrane regions are eliminated, and amino acid sequence represented by SEQ. ID. No 6 and comprising His6-tag amino acid sequence altogether (see FIG. 1), and transfected host cells with a-LAGT fusion gene encoding the above mentioned fusion protein, resulting in the confirmation of the expression of 62 kDa sized fusion protein (see FIG. 2). The present inventors further investigated about the activity of the fusion protein expressed in the above transformed cells. As a result, we, the present inventors, confirmed that the fusion protein had not only the activity of galactosyltransferase but also the activities of N-acetylgalactosaminyltransferase and lactosyltransferase at the same time (see FIG. 3). In addition, unlike the control group, the fusion protein expressed in the transformed cells of the present invention was confirmed to have the activity of glucosyltransferase (about 120-130 p mol/h) as well.

Therefore, the fusion protein of the present invention includes the activities of four glycotransferases such as galactosyltransferase, N- acetylgalactosaminyltransferase, lactosyltransferase and glucosyltransferase, which were not included in existing proteins except the activity of galactosyltransferase.

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

It is preferable for the gene encoding the fusion protein of the present invention to comprise the gene represented by SEQ. ID. No 1 and encoding a- lactalbumin and the gene represented by SEQ. ID. No 3 and encoding j3 1, 4-galactosyltransferase-1. The gene encoding the fusion protein of the present invention can additionally have the genes encoding restriction enzyme recognition sequence, a non-functional linker and His6-tag.

It is also preferable for the gene encoding the fusion protein of the present invention to comprise the gene represented by SEQ. ID. No 1 and encoding a- lactalbumin, the gene represented by SEQ. ID. No 3 and encoding P 1, 4-galactosyltransferase-1 wherein cytosolic tail and transmembrane domain are removed and the gene represented by SEQ. ID. No 5 and containing restriction enzyme recognition sequence between the two said genes. However, sequences that can be included in the gene of the present invention are not limited thereto, yet genes encoding every fusion protein that has the activity of N-acetylgalactosaminyltransferase can be included.

In the preferred embodiments of the present

invention, the present inventors combined the gene encoding a-lactalbumin with the gene encoding j3 1,4- galactosyltransferase-1 wherein cytosolic tail and transmembrane domain were absent using a non-functional linker, a base sequence corresponding to two amino acid (Leu-Glu), and prepared a fusion protein represented by SEQ. ID. No 5 by adding a base sequence corresponding to His6-tag to the end of the gene encoding P 1,4- galactosyltransferase-1. The present inventors named the prepared fusion protein"a-LAGT".

The present invention further provides transformed cells transfected with the above gene.

In the preferred embodiments of the present invention, the present inventors induced the expression of SV40 large-T antigen using Lec8 cells in which galactosylation is deficient in their glycoprotein, leading to the preparation of Lec8-T single cells. The Lec8-T single cells can express transfected proteins continuously and are useful for detecting the neo- glycosylation, such as lacdiNAcylation. The present inventors, then, transfected the above cells with a- LAGT fusion gene represented by SEQ. ID. No 5 by liposome-mediated gene transfection, by which cells expressing fusion proteins continuously were obtained.

The present inventors confirmed that 62 ka-sized

fusion protein was expressed in the transformed cells transfected with a-LAGT fusion protein (see FIG. 2).

The present inventors also investigated the activity of the fusion protein expressed in the transformed cells, resulting in the confirmation that the fusion protein had the activities of galactosyltransferase, N- acetylgalactosaminyltransferase and lactosyltransferase at the same time (see FIG. 3). In addition, the present inventors further confirmed that the fusion protein expressed in the transformed cells also had glucosyltransferase activity (about 120-130 p mol/h).

Additionally, the present inventors confirmed that the transformed cells expressing the fusion protein of the present invention produced a protein comprising LacdiNAc-R (GalNAc-P 1,4-GlcNAc-R) glycoprotein structure actually in vivo (see FIG. 4).

Therefore, the fusion protein produced in the transformed cells of the present invention by combining a-lactalbumin with P 1, 4-galactosyltransferase-1 has the activity of N-acetylgalactosaminyltransferase which is absent in a-lactalbumin and P 1,4- galactosyltransferase-1. The fusion protein can produce a protein comprising LacdiNAc-R glycoprotein structure owing to the activity of N- acetylgalactosaminyltransferase. In addition to the activity of N-acetylgalactosaminyltransferase, the

fusion protein produced in the transformed cells of the present invention also contains the activities of lactosyltransferase, glucosyltransferase and galactosyltransferase.

The above mentioned a-LAGT fusion gene was constructed by the present inventors for the first time and no cell line that is available for the preparation of single polypeptide chain protein comprising LacdiNAc-R glycoprotein structure and having the activity of N-acetylgalactosaminyltransferase has been reported yet. Thus, the present inventors developed the said transformed cell line and named it"Lec8/LG60", which was deposited at Gene Bank of Korea Research Institute of Bioscience and Biotechnology on April 12, 2002 (Accession No : KCTC 10223BP).

The present invention also provides a preparation method for producing a recombinant protein comprising LacdiNAc-R glycoprotein structure by using the above transformed cell line.

The preparation method of the present invention for producing a protein having LacdiNAc-R glycoprotein structure comprises the following steps: 1) Constructing expression vector by inserting a gene encoding a target protein therein; 2) Transfecting the transformed cells of the

present invention with the expression vector of the above step 1); 3) Culturing the transfected cells of the above step 2); and 4) Separating and purifying the target protein from the cell culture solution of the above step 3).

The above mentioned gene encoding a target protein implies the gene encoding glycoprotein forming LacdiNAc-R structure during protein synthesizing process and is exemplified by genes encoding lutenizing hormone, FSH (follicle stimulating hormone), thyrotrophins, urokinase, protein C and glycodelin, etc.

Besides, every gene encoding glycoprotein forming LacdiNAc-R structure can be included in the category of the above-mentioned gene.

Conventional methods for introducing gene, cell culture, and separating and purifying a target protein that are well known to every person in this field were used for introducing a target gene into the transformed cells expressing the fusion protein of the present invention, for culturing thereof in order to mass- express the target protein and for separating and purifying the target protein massively expressed and secreted in the cell culture solution.

The transformed cells of the present invention

produce fusion proteins in which a-lactalbumin and j3 1/4-galactosyltransferase-1 are combined. The fusion protein has the activity of N- acetylgalactosaminyltransferase that is absent in a- lactalbumin and ß 1/4-galactosyltransferase-1. Owing to the activity, a protein comprising LacdiNAc-R glycoprotein structure is synthesized. Therefore, a target protein having LacdiNAc-R structure can be produced by introducing a target gene into the transformed cells of the present invention that is available for the production of a protein having LacdiNAc-R structure with the activity of N- acetylgalactosaminyltransferase.

BRIEF DESCRIPTION OF THE DRAWINGS The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein: FIG. 1 is a diagram showing the structure of 1/4-galactosyltransferase-1 (up) and the structure of a-LAGT fusion gene of the present invention (down); Gal-T-1 : Whole gene of ß 1/4- galactosyltransferase-1, s-Gal-T-1 : Partial gene of secreted type ß 1/4-

galactosyltransferase-1, cy : Cytosolic tail, m : Transmembrane domain, stem : Stem region, c : Catalytic domain, s : Signal sequence, Signal sequence cleavage site, FIG. 2 is a photograph showing the result of Western blot analysis with fusion proteins expressed in transformed cells after a-LAGT fusion gene was introduced in Lec8-T cells for transformation; FIG. 3 is a graph showing the activities of galactosyltransferase (gal-T), lactosyltransferase (lac-T) and N-acetylgalactosaminyltransferase (lacdiNAc-T) of the fusion protein of the present invention ; FIG. 4 is a photograph showing the result of Western blot analysis using LacdiNAc-R specific monoclonal antibody, which was performed to investigate whether the fusion protein of the present invention forms LacdiNAc-R structure.

EXAMPLES Practical and presently preferred embodiments of

the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Preparation of a-LAGT fusion gene by combining a-lactalbumin cDNA with ß 1/4- galactosyltransferase cDNA In order to prepare a fusion protein having the activity of N-acetylgalactosaminyltransferase, the present inventors prepared a fusion gene by combining whole cDNA (SEQ. ID. No 1; GenBank Accession # : J05147) encoding bovine a-lactalbumin with whole cDNA (SEQ. ID.

No 3; GenBank Accession # : X55415) encoding human 1, 4-galactosyltransferase-1.

Particularly, the present inventors prepared a fusion gene by combining whole cDNA represented by SEQ.

ID. No 1 and encoding bovine a-lactalbumin with modified P 1/4-galactosyltransferase (Arg49-Ser398) gene in which 48 amino acids of NH2 terminal (cytosolic tail and transmembrane domain) were removed at the ratio of 1: 1. Induced secretion of a fusion protein

produced by the expression of the fusion gene using signal sequence of a-lactalbumin. In other words, the secretion of the fusion protein of the present invention in culture medium was caused by signal sequence of a-lactalbumin included in the fusion gene and then the signal sequence of a-lactalbumin (lut- l9th). was cut off.

As shown in FIG. 1, the present inventors added two amino acids (Leu-Glu) to the combining region of a-lactalbumin and J3 1, 4-galactosyltransferase gene as a non-functional linker, and then added His6-tag to the end of j3 1, 4-galactosyltransferase. The present inventors named the prepared fusion gene"a-LAGT" (FIG.

1).

Example 2: Preparation of transformed cell lines expressing fusion proteins The present inventors prepared transformed cell lines that can continuously express the fusion gene constructed in the above Example 1.

Particularly, used Lec8 cell line (ATCC, #CRL1737) as a host cell line for transduction and induced the expression of SV40 large-T antigen therein

for the continuous high expression of a-LAGT fusion gene that was to be transfected into the cell line.

The present inventors named the cell line"Lec8-T".

More precisely, transfected Lec8 cell line with a gene expressing SV40 large-T antigen (Choi C. S et al., Arch.

Virol., 1990, 115 (3-4), 227-37 ; Tang W. J. and Folk W. R., J Virol, 1989, 63 (12), 5478-82 ; de Chasseval R and de Villartay J. P., Nucleic Acids Res, 1992, 20 (2), 245-50) by liposome-mediated gene-transfection and cultured thereof in a medium containing G418 antibiotics (400 fla /m. C). During the culture, selected living cells, leading to the preparation of stable single clones expressing the large T-antigen continuously.

Once a-LAGT fusion gene is introduced in Lec8-T cells, the gene can be expressed highly and continuously. The present inventors inserted a-LAGT fusion gene prepared in the above Example 1 into pcDNA6 expression vector (Invitrogen), with which Lec8-T cells were transfected by using liposome-mediated gene transfection. As a result, stable single cells expressing a-LAGT fusion gene continuously were obtained and named"Lec8/LG10","Lec8/LG35"and "LecB/LG60"each. At the same time, cells transfected with pcDNA6 vector in which the fusion gene was not inserted were prepared for control and named"Lec8

mock".

Example 3: Analysis on the expression of fusion protein in transformed cells In order to confirm whether the transformed cells prepared in the above Example 2 could express the inserted fusion protein, the present inventors performed Western blotting. For the pure separation, the fusion protein of the present invention was prepared by attaching His6-tag to the COOH-end of ß 1, 4-galactosyltransferase gene. Thus, the expressed fusion protein was easily separated by using Ni-NTA- agarose affinity column. The expression level of the secreted fusion protein was analyzed by Western blotting using a-lactalbumin specific antibody.

Particularly, the cells transfected with a vector containing the fusion protein of the present invention and the control cells (Lec8 mock) transfected with a vector without the fusion protein were both washed with ix PBS buffer (6.7 mM KH2PO4/150 mM NaCl/pH 7. 4), and then dissolved using PBS buffer containing 0. 1% NP-40.

Total cell proteins were separated by SDS-PAGE electrophoresis. The separated proteins were transferred onto nitrocellulose membrane, after which

ECL (Enhanced chemiluminescence, Amersham) was performed using rabbit a-lactalbumin specific antibody (1: 500 dilution) (Bethyl laboratories) as the primary antibody and anti-rabbit IgG-peroxidase (1: 4000 dilution) (Sigma) as the secondary antibody.

As a result, the transformed cells Lec8/LG10, Lec8/LG35 and Lec8/LG60 expressing a-LAGT fusion gene expressed 62 kDa fusion protein, and among them, Lec8/LG60 expressed the fusion protein of the present invention most (FIG. 2).

Example 4: Measurement of the enzyme activity of the fusion protein In order to investigate the enzyme activity of the fusion protein expressed in the transformed cells prepared in the above Example 2, the present inventors separated the fusion protein from the transformed cells, followed by the measurement of the activities of galactosyltransferase, lactosyltransferase and N- acetylgalactosaminyltransferase.

Particularly, pure separation of the fusion protein secreted in the cell culture medium was carried out by using Ni-NTA-agarose. The separated fusion

proteins were then washed with lX PBS buffer. The enzyme activity was measured by taking advantage of Ni- bead that was combined with the fusion protein. Used 20 mM GlcNAc (Sigma) and 0.1 u Ci [6-3H] UDP-galactose (New England Nuclear) as substrates for the total reaction volume of 50/and loaded Ni-bead combined with the fusion protein into 100 mM Na-cacodylate buffer (pH 7.0) (Sigma) containing 20 mM MnCl2, followed by reacting at 37C for 2 hours. Let the reaction product pass through Dowex lx 8 (Cl-form) column (BioRad) and Sepharose 4B column (BioRad) to measure the cpm value of the reaction product. At last, compared the enzyme activities.

For the measurement of enzyme activity of lactosyltransferase, used [6-3H] UDP-galactose and 20 mM glucose as substrates under the same experimental conditions as above. For the measurement of enzyme activity of N-acetylgalactosaminyltransferase, used [6- 3H] UDP-GalNAc (New England Nuclear) as a donor substrate and GlcNAc as an acceptor substrate.

As a result, the fusion protein of the present invention was proved to have all the activities of galactosyltransferase, N- acetylgalactosaminyltransferase and lactosyltransferase (FIG. 3).

The fusion protein was loaded in 20 yt of reaction buffer (ATP 5 mM, GlcNAc 20 mM, UDP- [3H] Glucose (1X 105 cpm) 100 g M/MnCl2 20 mM/sodium cacodylate buffer 100 mM, pH 6. 7), followed by reacting at 37C for 2 hours.

1 mt of cold distilled water was added to stop the reaction. The reaction product was enforced to pass through 1.0 mt column (Dowex AG 1-X8 (Cl-form)) and the fusion protein was separated by using Dowex lx 8 (Cl- form) column. The cpm value of the separated fusion protein was measured to detect the enzyme activity thereof.

As a result, the fusion protein of the present invention was confirmed to have the activity of glucosyltransferase by 120-130 pmol/hour.

Example 5: Analysis on the formation of LacdiNAc-R glycoprotein structure of the fusion protein In order to investigate whether the fusion protein of the present invention was really forming LacdiNAc-R (GalNAc-J3 1, 4-GlcNAc-R) glycoprotein structure, the present inventors analyzed the activity of the fusion protein using LacdiNAc-R glycoprotein- specific monoclonal antibody.

Particularly, cultured Lec8/LG60, Lec8/LG35 and Lec8/LG10 cell lines having the N- acetylgalactosaminyltransferase activity and expressing the fusion protein of the present invention along with control cells in media (alpha-MEM+10% fetal bovine serum, Gibco BRL) containing G418 (400 ßg/mQ) (Gibco BRL) and blasticidine (10 Ag/mQ) (Invitrogen). Washed the cultured cells with lx PBS buffer, followed by sonication in 20 mM Na-cacodylate buffer (pH 7. 0) containing 1 mM CaCl2 in order to remove nuclei.

Carried out ultracentrifugation at 100, 000 g for 1 hour, after which separated microsome. SDS-PAGE electrophoresis with membrane glycoproteins was performed, which were transferred onto nitrocellulose membrane. ECL (Enhanced chemiluminescence) was finally performed using LacdiNAc-R-specific monoclonal antibody (1: 1000 dilution) (Nyame, A. K. et al., J. Parasitol, 2002, 88 (5), 890-7) as the primary antibody and anti- mouse IgM-peroxidase (1: 4000) (Sigma) as the secondary antibody (exposed with Kodak BioMax MR1 film).

As a result, monoclonal antibody reaction responding to LacdiNAc-R glycoprotein structure was not observed in control cells while the strong reaction was detected in the transformed cells of the present invention (FIG. 4), suggesting that those transformed

cells expressing the fusion protein of the present invention had the activity of N- acetylgalactosaminyltransferase and, at the same time, produced proteins having LacdiNAc-R glycoprotein structure. The present inventors deposited Lec8/LG60 that was the cell line expressing the fusion protein most at Gene Bank of Korea Research Institute of Bioscience and Biotechnology on April 12, 2002 (Accession No : KCTC 10223BP).

INDUSTRIAL APPLICABILITY As explained hereinbefore, the fusion protein of the present invention prepared by combining a- lactalbumin with ß 1/4-galactosyltransferase-1 has not only the activity of galactosyltransferase but also the activities of lactosyltransferase, glucosyltransferase and N-acetylgalactosaminyltransferase especially forming LacdiNAc-R glycoprotein structure. Thus/the fusion protein of the present invention can be effectively used for the production of recombinant medicinal proteins containing LacdiNAc-R glycoprotein structure.

Those skilled in the art will appreciate that the

conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention.

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