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Biological Activity of Human Dimeric Hyperglycosylated Erythropoietin (dHGEPO) Fusion Proteins
Naidansuren, Purevjargal,Min, Kwan-Sik The Korean Society of Animal Reproduction 2010 Reproductive & developmental biology Vol.34 No.4
Erythropoietin (EPO) is a glycoprotein hormone secreted from primarily cells of the peritubular capillary endothelium of the kidney, and is responsible for the regulation of red blood cell production. We constructed and expressed dimeric cDNAs in Chinease hamster ovary (CHO) cells encoding a fusion protein consisting of 2 complete human EPO domains linked by a 2-amino acid linker (Ile-Asp). We described the activity of dimeric hyperglycosylated EPO (dHGEPO) mutants containing additional oligosaccharide chains and characterized the function of glycosylation. No dimeric proteins with mutation at the $105^{th}$ amino acid were found in the cell medium. Growth and differentiation of the human EPO-dependent leukemiae cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of dHGEPO proteins. MIT assay at 24 h increased due to the survival of F36E cells. The dHGEPO protein migrated as a broad band with an average molecular mass of 75 kDa. The mutant, dHGEPO, was slightly higher than the wild-type (WT) dimeri-EPO band. Enzymatic N-deglycosylation resulted in the formation of a narrow band with a molecular mass twice of that of of monomeric EPO digested with an N-glycosylation enzyme. Hematocrit values were remarkably increased in all treatment groups. Pharmacokinetic analysis was also affected when 2.5 IU of dHGEPO were intravenously injected into the tails of the mice. The biological activity and half-life of dHGEPO mutants were enhanced as compared to the corresponding items associated the WT dimeric EPO. These results suggest that recombinant dHGEPO may be attractive biological and therapeutic targets.
Naidansuren, Purevjargal,Min, Kwan-Sik The Korean Society of Animal Reproduction 2012 Reproductive & developmental biology Vol.36 No.2
We prepared the polyclonal antibody anti-$20{\alpha}$-hydroxysteroid dehydrogenase (anti-$20{\alpha}$-HSD) against the recombinant full-length protein bovine $20{\alpha}$-HSD in Escherichia coli. The specificity of anti-$20{\alpha}$-HSD was demonstrated using Chinese hamster ovary (CHO) cells transfected with recombinant bovine $20{\alpha}$-HSD and bovine placental tissues. According to western blot analysis, anti-$20{\alpha}$-HSD specifically recognizes the 37-kDa protein bovine $20{\alpha}$-HSD. The protein is not present in untransfected CHO cells. Anti-$20{\alpha}$-HSD also recognizes a specific protein in the ovaries and placenta of other animals. Immunostaining was used to detect expression of bovine $20{\alpha}$-HSD protein in the cultured luteal cells during the estrous cycle later.
Development and Characterization of Hyperglycosylated Recombinant Human Erythropoietin (HGEPO)
JarGal, Naidansuren,Min, Kwan-Sik The Korean Society of Animal Reproduction 2009 Reproductive & developmental biology Vol.33 No.2
Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. We constructed three EPO mutants ($\Delta$69, $\Delta$105 and $\Delta$69,105), containing an additional oligosaccharide chains. EPOWT and EPO$\Delta$69 were effectively expressed in transient and stably transfected CHO-K1 cell lines. But, it wasn't detected any protein in the culture medium of EPO$\Delta$105 and EPO$\Delta$69,105 mutants. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure the cytokine dependency and in vitro bioactivity of rec-hEPO. MTT assay values were increased by survival of F36E cells at 24h. To analysis biological activity in vivo, two groups of ICR-mice (7 weeks old) were injected subcutaneously with 10 IU per mice of rec-hEPO molecules on days 0 and 2. Red blood cell and hematocrit values were measured on 6 days after the first injection. The hematocrit values were remarkably increased in all treatment groups. The pharmacokinetic analysis was also affected in the mice injected with rec-hEPO molecules 2.5 IU by tail intravenous. Protein samples were detected by Western blotting. An EPO$\Delta$69 protein migrated as a broad band with an average apparent molecular and detected slightly high band. Enzymatic N-deglycosylation resulted in narrow band and was the same molecular size. The biological activity of EPO$\Delta$69 was enhanced to compare with wt-hEPO. The half-life was longer than wt-hEPO. The results suggest that hyperglycosyalted recombinant human erythropoietin (EPO$\Delta$69) may have important biological and therapeutic good points.
Purevjargal Naidansuren,Kwan-Sik Min 한국동물번식학회 2012 Reproductive & developmental biology Vol.36 No.2
We prepared the polyclonal antibody anti-20α-hydroxysteroid dehydrogenase (anti-20α-HSD) against the recombinant full-length protein bovine 20α-HSD in Escherichia coli. The specificity of anti-20α-HSD was demonstrated using Chinese hamster ovary (CHO) cells transfected with recombinant bovine 20α-HSD and bovine placental tissues. According to western blot analysis, anti-20α-HSD specifically recognizes the 37-kDa protein bovine 20α-HSD. The protein is not present in untransfected CHO cells. Anti-20α-HSD also recognizes a specific protein in the ovaries and placenta of other animals. Immunostaining was used to detect expression of bovine 20α-HSD protein in the cultured luteal cells during the estrous cycle later.