Production of complex human glycoproteins in yeast.
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.
Hamilton
Stephen R
SR
Thayer School of Engineering and the Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.
Bobrowicz
Piotr
P
Bobrowicz
Beata
B
Davidson
Robert C
RC
Li
Huijuan
H
Mitchell
Teresa
T
Nett
Juergen H
JH
Rausch
Sebastian
S
Stadheim
Terrance A
TA
Wischnewski
Harry
H
Wildt
Stefan
S
Gerngross
Tillman U
TU
D000818:Animals; D020134:Catalytic Domain; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005818:Genetic Engineering; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D019151:Peptide Library; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014170:Transformation, Genetic
D006023:Glycoproteins; D026901:Membrane Transport Proteins; D019151:Peptide Library; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C121945:UDP-N-acetylglucosamine transporter; D017351:N-Acetylglucosaminyltransferases; D008361:Mannosidases