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Science 29 August 2003:
Vol. 301. no. 5637, pp. 1244 - 1246
DOI: 10.1126/science.1088166
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Reports

Production of Complex Human Glycoproteins in Yeast

Stephen R. Hamilton,* Piotr Bobrowicz,* Beata Bobrowicz,* Robert C. Davidson,* Huijuan Li,* Teresa Mitchell,* Juergen H. Nett,* Sebastian Rausch,* Terrance A. Stadheim,* Harry Wischnewski,* Stefan Wildt,* Tillman U. Gerngross{dagger}

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.

Thayer School of Engineering and the Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.



* Present address: GlycoFi, Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA.

{dagger} To whom correspondence should be addressed. E-mail: tillman.gerngross{at}dartmouth.edu

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