Enhanced sialylation of recombinant erythropoietin in CHO cells by human glycosyltransferase expression.
Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human alpha2,3- sialyltransferase (alpha2,3-ST) and beta1,4-galactosyltransferase (beta1,4-GT) are responsible for terminal sialylation and galactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of alpha2,3-ST and beta1,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When alpha2,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1%. When alpha2,3-ST and beta1,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of alpha2,3- ST and beta1,4-GT is more effective than the expression of alpha2,3-ST alone. Coexpression of alpha2,3-ST and beta1,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of alpha2,3-ST and beta1,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.
Jeong
Yeon Tae
YT
Department of Biological Sciences, KAIST, Daejeon 305-701, Korea.
Choi
One
O
Lim
Hye Rim
HR
Son
Young Dok
YD
Kim
Hong Jin
HJ
Kim
Jung Hoe
JH
D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D009237:N-Acetyllactosamine Synthase / Q000235:genetics / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011994:Recombinant Proteins; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D011994:Recombinant Proteins; D004921:Erythropoietin; D009237:N-Acetyllactosamine Synthase; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase