Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris.
Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-alpha-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of alpha-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.
Callewaert
N
N
Unit of Fundamental and Applied Molecular Biology, Department of Molecular Biology, Ghent University, Ghent, Belgium.
Laroy
W
W
Cadirgi
H
H
Geysens
S
S
Saelens
X
X
Min Jou
W
W
Contreras
R
R
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics; D009842:Oligopeptides; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D015202:Protein Engineering; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics*; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D006023:Glycoproteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D009842:Oligopeptides; D011134:Polysaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C077480:histidyl-aspartyl-glutamyl-leucine; D008361:Mannosidases; C069715:trans-sialidase; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D009439:Neuraminidase