title: Effect of mild-thiol reducing agents and alpha2,3-sialyltransferase expression on secretion and sialylation of recombinant EPO in CHO cells.
abstract: We have previously reported that N-acetylcysteine (NAC) not only delayed apoptosis but also enhanced the production of recombinant erythropoietin (EPO) in Chinese hamster ovary (CHO) cell culture. To investigate the production enhancement mechanism, the effects of similar thiolreducing agents were studied. Intriguingly, all mild reducing agents examined including mercaptoethanesulfonic acid (MESNA), thiolactic acid (TLA), and thioglycolate (TG) were shown to block apoptosis and increase EPO production. A pulse-chase study of EPO secretion revealed that all four thiol-reducing agents increased the EPO secretion rate; among them TLA showed the highest rate. In terms of product quality, the sialic acid content of the glycoprotein is one of the most important factors. It was reported that a number of glycoproteins produced by CHO cells often have incomplete sialylation, particularly under high-producing conditions. Human alpha2,3-sialyltransferase (alpha2,3-ST) was introduced into EPO-producing CHO cells in order to compensate for the reduced sialylation during supplementation with NAC. When alpha2,3-ST was expressed in the presence of NAC, reduced sialylation was restored and an even more sialylated EPO was produced. Thus, our study is significant in that it offers increased EPO production while still allowing the prevention of decreased sialylation of EPO.
authors: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
authors: Kern Hee
authors: KH
authors: Chang
authors: Yeon Tae
authors: YT
authors: Jeong
authors: Chan Yeong
authors: CY
authors: Kwak
authors: One
authors: O
authors: Choi
authors: Jung Hoe
authors: JH
authors: Kim
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006016:Glycolates / Q000378:metabolism; D006801:Humans; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism*; D055571:Secretory Pathway; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D006016:Glycolates; D011994:Recombinant Proteins; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D004921:Erythropoietin; D012799:Sialyltransferases; C023884:thiolactic acid