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

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.

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

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A Human Embryonic Kidney 293T Cell Line Mutated at the Golgi {alpha}-Mannosidase II Locus.

M. Crispin, V. T. Chang, D. J. Harvey, R. A. Dwek, E. J. Evans, D. I. Stuart, E. Y. Jones, J. M. Lord, R. A. Spooner, and S. J. Davis (2009)
J. Biol. Chem. 284, 21684-21695
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Dictyostelium discoideum--a promising expression system for the production of eukaryotic proteins.

R. Arya, A. Bhattacharya, and K. S. Saini (2008)
FASEB J 22, 4055-4066
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N-Glycan Modification in Aspergillus Species.

E. Kainz, A. Gallmetzer, C. Hatzl, J. H. Nett, H. Li, T. Schinko, R. Pachlinger, H. Berger, Y. Reyes-Dominguez, A. Bernreiter, et al. (2008)
Appl. Envir. Microbiol. 74, 1076-1086
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The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation.

S. Banerjee, P. Vishwanath, J. Cui, D. J. Kelleher, R. Gilmore, P. W. Robbins, and J. Samuelson (2007)
PNAS 104, 11676-11681
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Fucosylation in prokaryotes and eukaryotes.

B. Ma, J. L. Simala-Grant, and D. E. Taylor (2006)
Glycobiology 16, 158R-184R
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Humanization of yeast to produce complex terminally sialylated glycoproteins..

S. R. Hamilton, R. C. Davidson, N. Sethuraman, J. H. Nett, Y. Jiang, S. Rios, P. Bobrowicz, T. A. Stadheim, H. Li, B.-K. Choi, et al. (2006)
Science 313, 1441-1443
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Fucose Removal from Complex-Type Oligosaccharide Enhances the Antibody-Dependent Cellular Cytotoxicity of Single-Gene-Encoded Bispecific Antibody Comprising of Two Single-Chain Antibodies Linked to the Antibody Constant Region.

A. Natsume, M. Wakitani, N. Yamane-Ohnuki, E. Shoji-Hosaka, R. Niwa, K. Uchida, M. Satoh, and K. Shitara (2006)
J. Biochem. 140, 359-368
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A Model Vaccine Exploiting Fungal Mannosylation to Increase Antigen Immunogenicity.

J. S. Lam, M. K. Mansour, C. A. Specht, and S. M. Levitz (2005)
J. Immunol. 175, 7496-7503
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In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris.

W. Vervecken, V. Kaigorodov, N. Callewaert, S. Geysens, K. De Vusser, and R. Contreras (2004)
Appl. Envir. Microbiol. 70, 2639-2646
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