earticle

영어

The terminal sialylation of N-linked glycan is most important factor to determine the in vivo clearance rate of therapeutic glycoproteins. In this study, we introduced the sialuria-mutated rat GNE/MNK to increase the intracellular CMP-sialic acid by genetic engineering. From in vitro activity assays of various sialuria-mutated rat UDP-GlcNAc2-epimerases, rat GNE/MNK-R263L-R266Q mutant sustained the highest activity among the five mutants, and feedback inhibition was not detected. To improve the sialylation path way of CHO cells, Chinese hamster CMP-SAT, and human α2,3-ST were transfected simultaneously with sialuria-mutated rat GNE/MNK into the rhEPO-producing CHO cells. The intracellular CMP-sialic acid pool of engineered CHO cells was significantly increased up to 10.7 folds compared to control. The sialic acid contents of produced rhEPO were significantly increased up to 43%. The asialo-and monosialylated N-glycans were decreased by half compared to control, and the tetra-sialylated N-glycans were increased up to 32.0%. In this study, we found the effective sialuria-mutated rat GNE/MNK to increase the intracellular CMP-sialic acid pool by genetic engineering. And, the new CHO cell lines were constructed which could produce the more sialylated therapeutic glycoproteins by overexpression of sialuriamutated GNE/MNK, CMP-SAT, and α2,3-ST.