Production of complex human glycoproteins in yeast |
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Authors: | Hamilton Stephen R Bobrowicz Piotr Bobrowicz Beata Davidson Robert C Li Huijuan Mitchell Teresa Nett Juergen H Rausch Sebastian Stadheim Terrance A Wischnewski Harry Wildt Stefan Gerngross Tillman U |
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Affiliation: | Thayer School of Engineering and the Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA. |
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Abstract: | We report the humanization of the glycosylation pathway in the yeast Pichia pastoris to secrete a human glycoprotein with uniform complex N-glycosylation. The process involved eliminating endogenous yeast glycosylation pathways, while properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5'-diphosphate (UDP)-N-acetylglucosamine transporter. Targeted localization of the enzymes enabled the generation of a synthetic in vivo glycosylation pathway, which produced the complex human N-glycan N-acetylglucosamine2-mannose3-N-acetylglucosamine2 (GlcNAc2Man3GlcNAc2). The ability to generate human glycoproteins with homogeneous N-glycan structures in a fungal host is a step toward producing therapeutic glycoproteins and could become a tool for elucidating the structure-function relation of glycoproteins. |
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