Improvement of glycosylation in insect cells with mammalian glycosyltransferases.
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.
-hang
Geen Dong
GD
Graduate Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
Chen
Chen-Ju
CJ
Lin
Chih-Yin
CY
Chen
Hsei-Chorn
HC
Chen
Hungwen
H
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006019:Glycolysis / Q000235:genetics*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D016695:Glycosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D009036:Moths / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis* / Q000235:genetics
D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; D016695:Glycosyltransferases