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    • 8. 发明授权
    • Method for preparing recombinant glycoproteins with high sialic acid content
    • 制备高唾液酸含量的重组糖蛋白的方法
    • US08940876B2
    • 2015-01-27
    • US13390671
    • 2011-02-01
    • Jung Hoe KimYoung Dok SonJin Young HwangYeon Tae Jeong
    • Jung Hoe KimYoung Dok SonJin Young HwangYeon Tae Jeong
    • C07K14/505C07K14/705C07K14/52C12P21/00C12N9/90C12N9/12C12N9/10
    • C07K14/505C07K14/524C07K14/705C12N9/1081C12N9/1205C12N9/90C12P21/005C12Y501/03014
    • 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.
    • 本公开涉及一种制备高唾液酸含量的重组糖蛋白的方法。 更具体地,对于通过仅通过亮氨酸取代位置263处的精氨酸或通过用谷氨酰胺进一步取代位置266处的精氨酸而诱发点突变的UDP-GlcNAc 2-差向异构酶/ ManNAc激酶(GNE / MNK)酶,不断维持差向异构酶活性, 并且其过表达的细胞经历细胞内胞苷酸单磷酸(CMP) - 唾液酸含量的增加,而不考虑CMP-唾液酸浓度。 特别是,由于在产生点突变诱导的GNE / MNK的糖蛋白(例如促红细胞生成素和血小板生成素)中,人α-2,3-唾液酸转移酶和CMP-唾液酸转运蛋白基因同时过表达,因此CMP- 糖蛋白中的唾液酸和唾液酸在细胞中增加,在产生唾液酸化重组糖蛋白的宿主细胞中过表达,上述三种基因可能用于制备具有增加的唾液酸含量的糖蛋白。
    • 10. 发明申请
    • METHOD FOR PREPARING RECOMBINANT GLYCOPROTEINS WITH HIGH SIALIC ACID CONTENT
    • 用高碱性内容物制备重组蛋白糖的方法
    • US20120149874A1
    • 2012-06-14
    • US13390671
    • 2011-02-01
    • Jung Hoe KimYoung Dok SonJin Young HwangYeon Tae Jeong
    • Jung Hoe KimYoung Dok SonJin Young HwangYeon Tae Jeong
    • C07K14/435C12N9/18C12N9/10C07K14/575C12N15/81C07K14/745C12N15/85C12N1/19C12N5/10C12P21/00C07K14/755
    • C07K14/505C07K14/524C07K14/705C12N9/1081C12N9/1205C12N9/90C12P21/005C12Y501/03014
    • 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.
    • 本公开涉及一种制备高唾液酸含量的重组糖蛋白的方法。 更具体地,对于通过仅通过亮氨酸取代位置263处的精氨酸或通过用谷氨酰胺进一步取代位置266处的精氨酸而诱发点突变的UDP-GlcNAc 2-差向异构酶/ ManNAc激酶(GNE / MNK)酶,不断维持差向异构酶活性, 并且其过表达的细胞经历细胞内胞苷酸单磷酸(CMP) - 唾液酸含量的增加,而不考虑CMP-唾液酸浓度。 特别是,由于在产生点突变诱导的GNE / MNK的糖蛋白(例如促红细胞生成素和血小板生成素)中,人α-2,3-唾液酸转移酶和CMP-唾液酸转运蛋白基因同时过表达,因此CMP- 糖蛋白中的唾液酸和唾液酸在细胞中增加,在产生唾液酸化重组糖蛋白的宿主细胞中过表达,上述三种基因可能用于制备具有增加的唾液酸含量的糖蛋白。