Improvement of glycosylation in insect cells with mammalian glycosyltransferases |
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Authors: | -hang Geen Dong Chen Chen-Ju Lin Chih-Yin Chen Hsei-Chorn Chen Hungwen |
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Affiliation: | Graduate Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan. |
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Abstract: | The N-glycans of recombinant glycoproteins expressed in insect cells mainly contain high mannose or tri-mannose structures, which are truncated forms of the sialylated N-glycans found in mammalian cells. Because asialylated glycoproteins have a shorter half-life in blood circulation, we investigated if sialylated therapeutic glycoprotein can be produced from insect cells by enhancing the N-glycosylation machinery of the cells. We co-expressed in two insect cell lines, Sf9 and Ea4, the human alpha1-antitrypsin (halpha1AT) protein with a series of key glycosyltransferases, including GlcNAc transferase II (GnT2), beta1,4-galactosyltransferase (beta14GT), and alpha2,6-sialyltransferase (alpha26ST) by a single recombinant baculovirus. We demonstrated that the enhancement of N-glycosylation is cell type-dependent and is more efficient in Ea4 than Sf9 cells. Glycan analysis indicated that sialylated halpha1AT proteins were produced in Ea4 insect cells expressing the above-mentioned exogenous glycosyltransferases. Therefore, our expression strategy may simplify the production of humanized therapeutic glycoproteins by improving the N-glycosylation pathway in specific insect cells, with an ensemble of exogenous glycosyltransferases in a single recombinant baculovirus. |
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