Enhanced sialylation of recombinant erythropoietin in genetically engineered Chinese-hamster ovary cells.
Sialic acid, the terminal sugar in N-linked complex glycans, is usually found in glycoproteins and plays a major role in determining the circulatory lifespan of glycoproteins. In the present study we attempted to enhance the sialylation of recombinant EPO (erythropoietin) in CHO (Chinese-hamster ovary) cells. To enhance EPO sialylation, we introduced human alpha2,3-ST (alpha2,3-sialyltransferase) and CMP-SAS (CMP-sialic acid synthase) into recombinant human EPO-producing CHO cells. The sialylation of EPO was increased by the expression of alpha2,3-ST alone. Although the co-expression of alpha2,3-ST and CMP-SAS did not further increase sialylation, an increase in the intracellular pool of CMP-sialic acid was noted. On the basis of these observations, it was postulated that the transport capacity of CMP-sialic acid into the Golgi lumen was limited, thereby causing the reduced availability of CMP-sialic acid substrate for sialylation. Therefore, we co-expressed human alpha2,3-ST and CMP-SAS, as well as overexpress Chinese hamster CMP-sialic acid transporter (CMP-SAT) in CHO cells, which produced recombinant human EPO. When alpha2,3-ST, CMP-SAS, and CMP-SAT were overexpressed in CHO cells, there was a corresponding increase in sialylation compared with the co-expression of alpha2,3-ST and CMP-SAS. The present study provides a useful strategy for enhancing the sialylation of therapeutic glycoproteins produced in CHO cells.
Jeong
Yeon Tae
YT
Department of Biological Sciences, KAIST (Korea Advanced Institute of Science and Technology), 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Choi
One
O
Son
Young Dok
YD
Park
Seung Yeol
SY
Kim
Jung Hoe
JH
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid