KR100604258B1 - Method for enhancing in vivo activity of glycoproteins - Google Patents

Abstract

The present invention minimizes the production of sialidase by modifying the sialidase gene of a cell line producing glycoprotein, and uses a specific enzyme for a glycoprotein in which sialic acid is released from the glycoprotein. By recombining sialic acid, the present invention relates to a method for maximizing the content of sialic acid in a glycoprotein to increase the in vivo activity of the glycoprotein.

Description

Method for enhancing in vivo activity of glycoproteins

1 shows the cloning of Sial36 and Sial78; And

Figure 2 is a schematic of the vector (pKO-ZDSial7836) used for causing homologous recombination in the present invention.

The present invention relates to a method for increasing the in vivo activity of glycoproteins by maximizing the content of sialic acid in glycoproteins. More specifically, the present invention minimizes the production of sialidase of glycoprotein-producing cell lines, while sialic acid recombines sialic acid with a specific enzyme to the glycoprotein from which sialic acid is released, thereby sialic acid possessed by the glycoprotein. The present invention relates to a method for increasing the in vivo activity of glycoproteins.

Glycoprotein is one of the many sugar compounds in nature, and has a structure in which one or more oligosaccharides are linked to a polypeptide chain linked by amino acids. This glycostructure affects the properties, solubility, longevity and activity of the protein. Glycosylation of proteins is not directly regulated by genes but occurs by modification of various enzymes after the translation of the protein is completed. Some of these glycoproteins can be artificially produced in large quantities by biotechnological methods and used as valuable medicines for humans.

Usually oligosaccharides bind to asparagine, serine or threonine in proteins, or sometimes to the carboxy terminus. The sugar structure that binds to asparagine in the protein is called N-linked sugar structure, and has a common N-acetyl glucosamine (pentasaccharide core) consisting of two N-acetyl glucosamine and three mannose. The structure formed by further combining several mannoses with the five-saccharide structure is called an oligo-mannose type sugar structure, and the structure formed by further combining N-acetyl lactosamine units is complex type ( complex type) or N-acetyl lactosamine type. In addition, the mannose structure and the lactosamine structure coupled to the pentose structure is called a hybrid type (hybrid type). Complex oligosaccharides may have several branches, usually two to five branches, and may be combined with sialic acid, galactose, or fucose at their ends.

Sialic acid is a generic term for several types of neuramic acid, and sialic acid commonly found in glycoproteins is N-acetyl neuraminic acid (Neu5Ac) or N-glycosyl neuramic acid. (N-glycosyl neuraminic acid (Neu5Gc)), usually with α2-3 or α2-6 bonds, with non-sugar substances such as sulfate, phosphate, 2-aminoethyl phosphate, alkyl, and acyl groups. .

In the case of O-linked sugar chains in which the oligosaccharide is bonded to serine or threonine in the polypeptide chain, there is only a mucin type bound by N-acetyl galactosamine (GalNAc). Gal, GlcNAc, GalNAc or sialic acid bind to GalNAc in turn. Glycoprotein oligosaccharides are formed by elements other than proteins or nucleic acids. For example, the polypeptide tertiary structure around which the oligosaccharides are bound, the activity of the modifying enzyme, the concentration of sugar donors, or the enzyme's access The possible structure determines the final structure of the sugar chains. Normally, N-linked sugar chains are bound to Asn-X-Ser / Thr sequences in a polypeptide, with only about one third of the possible Asn-X-Ser / Thr sequences actually binding to sugar chains. Sometimes the Asn-X-Cys sequence binds to sugar chains. The number of branches added to the sugar chain is determined by the activity of N-acetylglucosaminyl transferase. Mucin-type O-linked sugar chains are produced in smooth endoplasmic reticulum and Golgi bodies.

The shape of the oligosaccharides is different depending on the type of cell. Expression of the same glycoprotein in different cell types results in distinct oligosaccharide forms. Bacterial cells have been known to not form oligosaccharides in proteins, but recently, some types of bacterial cells have been found to form oligosaccharides. In yeast cells glycosylation occurs and has O- and N-linked sugar chains comprising mannose. Since insect cells do not have β1-4 galactosyl transferase activity, most have only mannose or GlcNAc at the end of the N-linked sugar chain. When producing recombinant glycoproteins using plant cells, it is found that β1-2 bound xylose or α1-3 bound fucose are bound to the 5-glycostructure.

Glycosylation may be affected by various environmental factors during cell culture. Usually, abnormally shortened sugar chains are combined, and the concentration of sugar donor is low, so that only a part of the protein is glycosylated, thus affecting the tertiary structure or solubility of the protein. It's crazy. The effects of glucose in the medium are different depending on the type of cells, and these effects are usually reduced by the addition of mannose, but the addition of fructose, galactose, inositol, pyruvate or glutamine has little effect. have. Cell density or growth rate may affect the production of Glc ₃ Man ₉ GlcNAc ₂ precursors, thereby affecting glycosylation of proteins. Reagents such as hormones, dimethylsulfoxide, phorbol esters or retinoic acid act on transcription and affect the concentration of glycosylation enzymes such as glycosyl transferase or glycosidase. Hormones interfere with vesicle trafficking, which prevents the glycoprotein from transferring from the rough endoplasmic reticulum to the Golgi apparatus. Ammonia is a byproduct of amino acid metabolism that affects not only cell growth and cell activity but also glycosylation of proteins. Amine or EDTA used in the medium affects vesicle trafficking by changing pH or ion concentration.

In vivo activity of glycoproteins is highly correlated with the presence or absence of sialic acid at the end of the sugar chain structure. In other words, when sialic acid is removed from glycoproteins, the in vivo activity of glycoproteins is lost. This is because sialic acid is dropped from glycoproteins, and galactose exposed in glycoproteins binds to the hepatocyte galactose binding protein. This is because glycoproteins are rapidly broken down in the liver.

Many animal cells that produce glycoproteins have intracellular sialidases, which can separate sialic acid from the sugar chain portion of the glycoprotein. In animal cell culture for glycoprotein production, glycoproteins are released outside the cell and thus are not expected to be affected by intracellular sialidase, but in practice a large number of sialic acids are released from glycoproteins. According to recent literature, the release of sialidase from animal cells out of cells is reported to be free from the cytoplasm of cells that die during batch culture. Therefore, it can be observed that the sialidase activity in the culture medium gradually increases during the batch culture of the animal cells for a long time.

Several methods are known for protecting glycoproteins from sialidases, but the most fundamental method would be to natively inactivate sialidase in animal cells.

The inventors of the present invention have made it possible to inactivate specific genes in cell chromosomes due to the recent development of genetic engineering techniques, and thus the present invention can be applied to inactivate the sialidase genes in cells producing glycoproteins. And that sialic acid may be freed using specific enzymes to recombine sialic acid to exposed galactose, maximizing the content of sialic acid in the glycoprotein to increase the in vivo activity of the glycoprotein. As a result of continuous research to develop a new method, the present invention has been completed.

Accordingly, an object of the present invention is a method of preparing a glycoprotein-producing cell line in which the intracellular sialidase titer is lowered by inactivating the sialidase gene in the cellular chromosome, the intracellular sialidase titer prepared by the above method. It is to provide a reduced glycoprotein-producing cell line, and a method for producing a glycoprotein with an increased in vivo titer of culturing the cell line to obtain a glycoprotein.

Another object of the present invention is to provide a method for producing a glycoprotein having increased activity in vivo by recombining sialic acid using a specific enzyme to a glycoprotein free of sialic acid in the glycoprotein prepared by the above method. will be.

In order to achieve the above object of the present invention, firstly, the present invention introduces an inactivated sialidase gene into a cell to induce homologous recombination with the sialidase gene in the chromosome and the homologous recombination. The present invention relates to a method for producing a glycoprotein-producing cell line with reduced intracellular sialidase titer, characterized by selecting cells inactivated by intracellular sialidase. In the method according to the invention, the inactivated sialidase gene is preferably prepared by inserting a selectable marker in the cloned sialidase gene.

Secondly, the present invention relates to a glycoprotein-producing cell line with lowered intracellular lipidase titer, which is prepared by the above method.

Third, the present invention relates to a method for producing glycoprotein with increased in vivo activity, wherein the cell line is cultured to obtain a glycoprotein.

Fourth, the present invention is characterized by recombination of sialic acid using a sialyl transferase to a glycoprotein free of sialic acid in the glycoprotein prepared by the above method. It relates to a method for producing a glycoprotein. In the method according to the invention, the sialic acid is preferably in the form of CMP-sialic acid formed from sialic acid and CTP by CMP-sialic acid synthetase.

In the methods according to the invention, it is also preferred that the glycoprotein is selected from the group consisting of erythropoietin, follicle gonadotropin (FSH) and tissue plasminogen activator (tPA).

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for producing a glycoprotein with increased in vivo activity by maximizing the content of sialic acid in the isolated and purified glycoprotein. That is, the present invention relates to a method of minimizing the production of sialidase of a cell line producing glycoprotein and recombining sialic acid using a specific enzyme to the glycoprotein in which the sialic acid is released from the produced glycoprotein.

According to the present invention, the sialidase gene of a glycoprotein producing cell line is first cloned (FIG. 1) and inactivated by inserting an identification marker. As such, a heterologous reporter sequence is inserted into the sialidase gene to inhibit the expression of the enzyme. After insertion into a suitable vector, the fragment is introduced into a glycoprotein producing cell line to induce homologous recombination with the sialidase gene in the chromosome. Sialidase-inactivated cell lines are selected through analysis of the identification gene and sialidase activity.

In addition, the present invention takes advantage of the fact that sialyl transferase can reattach sialic acid to galactose exposed by freeing sialic acid, thereby increasing the content of sialic acid by using sialyl transferase to the purified glycoprotein. By increasing the yield and low in vivo titer (low content of sialic acid), the discarded glycoproteins can be recovered through enzymatic reaction. Sialic acid must be combined with CMP (cytidine 5-monophosphate) to be used as a substrate for sialyl transferase, but since CMP-sialic acid is an expensive substance, it is recycled using CMP-sialic acid synthase from sialic acid and CTP. I tried to). The production of CTP from CMP requires the addition of phosphoenolpyruvate (PEP), pyruvate kinase (PK), myokinase (MK) and ATP. Since the reaction rate of the enzyme is proportional to the concentration of the substrate, it is advantageous to increase the concentration as long as the solubility and viscosity of the glycoprotein allow.

EXAMPLE

Hereinafter, the present invention will be described in more detail based on the preferred embodiments, which are only intended to aid the understanding of the present invention and are not intended to limit the scope of the present invention in any way.

Example 1 Isolation of Chromosome DNA

Chromosomal DNA was extracted as follows to isolate the sialidase gene from CHO cells.

After culturing CHO cells, 1 × 10 ⁸ cells Collected and washed twice with cold PBS. Cells were added to the centrifuge tube and filled with ice. After centrifugation at 500 ° C. for 10 minutes at 500 g, cells were suspended to 10 ⁷ cells / ml by adding PBS. To 10 ml of cell suspension was added 10 ml of cooled buffer C (320 mM sucrose, 5 mM MgCl ₂ , 10 mM Tris-HCl, 1% Triton X-100, pH 7.5) and 30 ml of cooled distilled water. After shaking, it was left for 10 minutes in ice. The mixture was centrifuged at 1300 g for 15 minutes at 4 ° C and the supernatant was removed. Again, 2 ml of the cooled buffer solution and 2 ml of the cooled distilled water were added thereto, shaken, and centrifuged at 4 ° C. and 1300 g for 15 minutes. 10 ml of buffer G2 (800 mM GuHCl, 30 mM EDTA, 30 mM Tris-HCl, 5% Tween-20, 0.5% Triton X-100, pH 8.0) was added and vigorously shaken for 10-30 seconds. 200 μl of proteinase K solution (20 mg / ml) was added thereto. After standing at 50 ° C. for 30 to 60 minutes, genomic DNA was purified using a Qiagene tip.

PCR was performed using the extracted DNA as a template. Two PCR products were obtained and each fragment was named "Sial36" {(2.3 Kb) and "Sial78" (562 bp). Sial36 includes a HindIII position at the 5 'end and a KpnI position at the 3' end, and Sial78 includes a BamHI position at the 5 'end and an EcoRI position at the 3' end. Treatment with restriction enzymes cloned sial36 and sial78 at the multicloning site in the pGEMT vector, respectively (see Figure 1). The Zeocine gene (1236 bp) was isolated from the pZeoSV2 (+) vector by treating the AscI enzyme, and the DT (Diphteria Toxin) gene (1184 bp) was processed by treating the pKO Select DT vector with RsrII. Each was isolated and inserted into the pGEMT vector. The completed pKO-ZDSial7836 vector is about 7247 bp in size. The construction of the vector as described above is shown in FIG.

Example 2 Transfection

After growing cells in a 60 mm cell culture dish to be 20-40% confluent (1-4 × 10 ⁵ cells / 60 mm dish), a pKO-ZDSial7836 plasmid was prepared (0.1 μg / μl or more). . 150 μl of the prepared plasmid DNA and cell medium (serum-free and antibiotic-free) were mixed well. 20 μl of Superfect Transfectin Reagent was added and mixed well. After 5-10 minutes of reaction at room temperature, the mixture was mixed well with 1 ml of cell medium. The mixture was added to a cell culture dish containing CHO cells and reacted at 37 ° C., 5% CO ₂ incubator for 2-3 hours. After washing four times with PBS, 5 ml of fresh medium was added. After culturing with medium replacement for 2-3 days, cells were split 1: 4 and zeocin was added to 50 μg / ml. The medium was replaced with zeocin-added medium for about 7 to 10 days, thereby killing both cells without the zeocin resistance gene and cells of the negative control.

Example 3 Screening of Sialidase Negative Cells

Cells selected in zeocin medium were cultured to 400 cells / 100 mm dishes and then the cell surface was covered with sterile Whatman paper. After incubation for about 11-14 days (medium change every 3-4 days), the paper was taken out, washed twice with PBS and dried. Paper was soaked in 0.1 mM 4MU NAcNeu (in 0.1 M sodium acetate) solution and allowed to react at room temperature for 1 hour. The paper was taken out and placed on a UV irradiator to observe fluorescence color and photographed (660 / 0.5 sec). The paper was dyed again by soaking in Coomassie Blue solution. Two photographs were compared to each other to select colonies that did not fluoresce. Corresponding colonies were separated from the original cell culture plates by trypsin treatment and then cultured separately.

Example 4 Sialylation of asialo-glycoprotein

As a buffer solution, 50 mM MOPS (4-morpholinepropanesulfonic acid) (pH 7.4) was used, and the reaction conditions were 30 ° C. and 20 to 40 hours.

Asialo-EPO (59.4 mg, 0.165 mmol) in PEP monopotassium salt (34 mg, 0.165 mmol), CMP (2.84 mg, 8.79 μmol), cilic acid (51 mg, 0.165 μmol) and ATP (0.532 mg, 0.879 μmol) ) Was added to pH 7.4, the final volume of 2.2 ml, and then MnCl ₂ was added to a final concentration of 30 mM. In addition, 13.2 units of myokinase and 19.8 units of pyruvate kinase (Boehringer Manheim) were added, and 1 unit of sialyl transferase was added to start the reaction at room temperature. Samples were obtained after 24 and 48 hours, respectively, and the degree of reaction was observed by IEF gel electrophoresis.

According to the present invention, by minimizing the production of sialidase of a cell line producing a glycoprotein and recombining the sialic acid using a specific enzyme to the glycoprotein in which the sialic acid is released from the glycoprotein, the sialic acid of the glycoprotein has By maximizing the content, it is possible to dramatically increase the in vivo activity of the glycoprotein.

Claims (7)

Inactivated sialidase genes are introduced into cells to induce homologous recombination with sialidase genes in the chromosome, and cultured cell lines in which intracellular sialidase is inactivated by the homologous recombination. To obtain a glycoprotein with increased activity in vivo; And Among the glycoproteins obtained in the previous step, comprising the step of recombining the sialic acid to the glycoprotein free of sialic acid using sialyl transferase and CMP- sialic acid, The CMP-sialic acid is formed from sialic acid and CTP by CMP-sialic acid synthetase (CMP-sialic acid synthetase) characterized in that the production of glycoproteins with increased activity in vivo. The method of claim 1, wherein the inactivated sialidase gene is prepared by inserting a selectable marker in the cloned sialidase gene. delete delete delete delete The method of claim 1 or 2, wherein the glycoprotein is selected from the group consisting of erythropoietin, follicle gonadotropin (FSH) and tissue plasminogen activator (tPA).

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