The overexpression of β-1,3-N-acetylglucosaminyltransferase 2(B3GNT2) and β-1,4-Galactosyltransferase 1 (B4GALT1) in Chinese hamster ovary cell
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Protein glycosylation is a post-translational modification that produces glycosides linkages through different types of metabolic pathways. Although the main structure of glycoprotein is defined by the amino acid sequence, its biological function and characteristic such as pharmacokinetics, hale-life, immunogenicity, solubility and stability can also be influenced by its oligosaccharide component. β-1,3-N-acetylglucosaminyltransferase(B3GNT2) and β-1,4-Galactosyltransferase (B4GALT1) contribute to the synthesis of LacNAc. We create single transfection and dual transfection of B3GNT2 and B4GALT1 into WT-EPO CHO cells and unsial-EPO CHO cells to detect their function on the extension of poly-LacNAc and determine whether those two glycotransferases can both overexpressed together. We created a set of engineered CHO cell lines: WT-EPO-B3GNT2, unsial-EPO-B3GNT2, WT-EPO-B4GALT1, unsial-EPO-B4GALT1, WT-EPO-B3GNT2-B4GALT1, unsial-EPO-B3GNT2-B4GALT1, WT-EPO-B4GALT1-B3GNT2 and unsial-EPO-B4GALT1-B3GNT2. We tested the transcriptional and translational overexpression levels of B3GNT2 and B4GALT1 in cell lines above. And We examined the EPO glycol profile from cell lines above. The result is those two genes cannot overexpress together and the single gene overexpression can make difference to the glycol profile but don’t make significant contribution to the extension of poly-LacNAc.