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Method for preparing recombinant glycoproteins with high sialic acid content

A technology of sialic acid and glycoprotein, applied in the field of preparing recombinant glycoprotein with high sialic acid content, which can solve the problems of shortening the life span of blood cells or glycoproteins

Active Publication Date: 2014-03-26
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the sialic acid of glycoconjugated glycans is located at the terminus of the glycan structure, which is present on the surface of the cell membrane, sialic acid was expected to be directly involved in the contact between the cell and the extracellular environment, and has recently been known to shorten the Increased lifespan of blood cells or glycoproteins in bodily fluids

Method used

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  • Method for preparing recombinant glycoproteins with high sialic acid content
  • Method for preparing recombinant glycoproteins with high sialic acid content
  • Method for preparing recombinant glycoproteins with high sialic acid content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Induction of Point Mutations in the UDP-GlcNAc 2-Epimerase / ManNAc Kinase (GNE / MNK) Gene

[0076] Substitution of amino acid 263 or amino acid 266 of GNE / MNK gene

[0077] by PCR using the forward primer (5'-ATGGAGAAGAACGGGAATAACCGG-3', SEQ ID NO: 6) and the reverse primer (5'-CTAGTGGATCCTGCGGGTCGTGTAG-3', SEQ ID NO: 7) from Rattus norvegicus The wild-type GNE / MNK gene (SEQ ID NO: 1) was amplified in liver tissue (obtained from Korea Advanced Institute of Science and Technology), and then, the primers for inducing point mutations and QuickChange Site-Directed Mutagenesis shown in Table 1 Kit (Stratagene), utilizing forward primer (5'-GGAGATGGTTCTAGTGATGCGAAG-3', SEQ ID NO:8) and reverse primer (5'-CCTCTACCAAGATCACTACGCCTTC-3', SEQ ID NO:9) for point mutation, Arginine at position 263 is replaced with leucine (SEQ ID NO: 2); or arginine at position 266 is replaced with glutamine or tryptophan.

[0078] Substitution of amino acid 263 or amino acid 266 of GNE...

Embodiment 2

[0087] Embodiment 2: Preparation of expression vector

[0088] Preparation of α-2,3-sialyltransferase expression vector

[0089] A forward primer (5'-ATGGGACTCTTGGT-3', SEQ ID NO: 14) and a reverse primer ( 5'-TCAGATGCCACTGCTTAG-3', SEQ ID NO: 15), human alpha-2,3-sialyltransferase was amplified from a human fibroblast cell line (HF cell line) by polymerase chain reaction (PCR). Such as image 3 As shown in , in order to introduce the amplified gene into Chinese hamster ovary cells as host cells, the amplified gene was ligated to the BamHI / HindIII site of the expression vector pcDNA3.1 / Zeo(+) to prepare the expression vector pcSTz( image 3 ).

[0090] Preparation of expression vector for cytidine monophosphate (CMP)-sialic acid transporter

[0091] A forward primer (5'-CAGCTAGCGCCACCATGGCTCAGG-3', SEQ ID NO: 16) and a reverse primer ( 5′-TCCGAATTCTCACACACCAATGACTC-3′, SEQ ID NO: 17), amplified by PCR from Chinese hamster ovary cells (EC2-1H9: obtained from Korea Resear...

Embodiment 3

[0094] Example 3: Identification of changes in sialic acid content caused by overexpression of GNE / MNK genes that induce point mutations

[0095] Introducing the expression vector of the GNE / MNK gene that induces point mutations into Chinese hamster ovary cells

[0096] By using Lipofectamine TM LTX and PLUS TM Reagent (Invitrogen), the vectors produced in Example were respectively transfected into Chinese hamster ovary cells, which are host cells producing recombinant erythropoietin. The transfected Chinese hamster ovary cells cultured in medium (MEM-α medium containing 400 μg / ml hygromycin B, 10% dFBS, 1% antibiotic-antimycotic, 20 nM MTX) were cultured in a culture medium with 5 %CO 2 Incubate in an incubator at 37°C, and repeat screening and subculture of surviving cells.

[0097] Identification of GNE / MNK gene expression

[0098] In order to identify the expression of the GNE / MNK gene introduced into the cells screened in Example , total RNA was isolated from the ...

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Abstract

The present disclosure relates to a method for preparing recombinant glycoproteins with high sialic acid content. More specifically, for UDP-GlcNAc 2-epimerase / ManNAc kinase (GNE / MNK) enzyme where point mutation was induced by substituting arginine at position 263 by leucine only or by further substituting arginine at position 266 by glutamine, epimerase activity is constantly maintained, and overexpressed cells thereof experience an increase in intracellular cytidine monophosphate (CMP)-sialic acid content, irrespective of CMP-sialic acid concentration. Particularly, since in an glycoprotein(such as, erythropoietin and thrombopoietin)-producing host cell where point mutationinduced GNE / MNK, human alpha-2,3-sialyltransferase and a CMP-sialic acid transporter gene are simultaneously overexpressed, intracellular content of CMP-sialic acid and sialic acid in glycoprotein increases in cells, overexpression in a host cell producing a sialylated recombinant glycoprotein the three genes above may be useful for preparing glycoprotein with increased sialic acid content.

Description

technical field [0001] The present application relates to methods of preparing recombinant glycoproteins with high sialic acid content. Background technique [0002] Sialic acid (Sia, NeuAc, NeuGc) is a general term for the acyl derivatives of neuraminic acid (Neu). Sialic acid, first isolated by Blix in 1936 from the mucin of the bovine salivary gland, is an acidic sugar containing 9 carbons with a COOH group. According to the difference of substituting groups, 50 kinds of sialic acids have been reported (Angata, T. and Varki, A., Chem.Rev, 102, 439-469, 2002), and these 50 kinds of sialic acids are known in different species and Tissue-specific distribution. In higher animals, sialic acid is connected to Gal, GlcNAc, GalNAc and sialic acid of glycoproteins, glycolipids and oligosaccharides through α-glycosidic bonds through the reaction of their respective specific sialyltransferases. [0003] Since the sialic acid of glycoconjugated glycans is located at the terminus o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/79C12N15/18C12P21/02
CPCC12P21/005C12N9/1205C07K14/705C12N9/90C07K14/524C12N9/1081C07K14/505C12Y501/03014C12N15/11C12N15/52C12N15/85
Inventor 金政会孙荣德黄真荣郑然太
Owner KOREA ADVANCED INST OF SCI & TECH
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