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.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Catalytic Domain
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Endoplasmic Reticulum / metabolism
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Genetic Engineering*
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Glycoproteins / biosynthesis*
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Glycoproteins / chemistry
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Glycoproteins / genetics
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Glycosylation
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Golgi Apparatus / metabolism
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Humans
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Mannosidases / genetics*
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Mannosidases / metabolism
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Membrane Transport Proteins / metabolism
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N-Acetylglucosaminyltransferases / metabolism
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Peptide Library
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Pichia / enzymology
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Pichia / genetics*
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Pichia / metabolism
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Polysaccharides / chemistry
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Polysaccharides / metabolism*
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Protein Processing, Post-Translational
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Protein Transport
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / biosynthesis*
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Transformation, Genetic
Substances
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Glycoproteins
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Membrane Transport Proteins
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Peptide Library
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Polysaccharides
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Recombinant Fusion Proteins
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Recombinant Proteins
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UDP-N-acetylglucosamine transporter
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N-Acetylglucosaminyltransferases
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Mannosidases