title: A functional analysis of N-glycosylation-related genes on sialylation of recombinant erythropoietin in six commonly used mammalian cell lines.
abstract: Significant efforts have been made to improve the sialylation of recombinant glycoproteins with the aim of extending their in vivo circulation time. Here, we report a systematic functional analysis of 31 N-glycosylation-related genes on sialylation of recombinant EPO in six cell lines. BHK and CHO cells were found to sialylate recombinant EPO most effectively. None of the 31 genes, individually or in combination, was able to improve EPO sialylation in these cells. HEK293, Cos-7 and 3T3 cells showed intermediate sialylation capabilities, whereas NS0 cells sialylated recombinant EPO poorly. Overexpression of ST6GalI, ST3GalIII or ST3GalIV, but not ST3GalVI, was able to improve EPO sialylation in these four cell lines. qRT-PCR experiments revealed that ST3GalIII and ST3GalIV are indeed under expressed in HEK293, 3T3 and NS0 cells. Co-expression of upstream glycogenes failed to synergize with these sialyltransferases to further enhance sialylation, suggesting that the upstream glycogenes are all expressed at sufficient levels.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros 138668, Singapore.
authors.forename: Peiqing
authors.initials: P
authors.lastname: Zhang
authors.forename: Diana Lifen
authors.initials: DL
authors.lastname: Tan
authors.forename: Desmond
authors.initials: D
authors.lastname: Heng
authors.forename: Tianhua
authors.initials: T
authors.lastname: Wang
authors.lastname: Mariati
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Yang
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Song
mesh_terms: D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007525:Isoelectric Focusing; D037102:Lectins / Q000737:chemistry; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012821:Silanes / Q000378:metabolism*; D017392:Thiobarbituric Acid Reactive Substances / Q000737:chemistry
chemical_list: D006023:Glycoproteins; D037102:Lectins; D011994:Recombinant Proteins; D012821:Silanes; D017392:Thiobarbituric Acid Reactive Substances; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid