title: Identification and removal of O-linked and non-covalently linked sugars from recombinant protein produced using Pichia pastoris.
abstract: The use of the methylotrophic yeast Pichia pastoris for large-scale recombinant production of proteins for therapeutic uses and/or biophysical characterisation has been gaining popularity. Here we describe the use of this organism for the production of a von Willebrand factor C domain from procollagen IIA for solution NMR studies. In this research, we specifically identified sites of O-linked glycosylation on the expressed protein, although the native protein is not glycosylated. We demonstrated that it was possible to remove the oligosaccharides by enzymatic digestion, however this approach proved to be prohibitively expensive for the scale of production required for high-resolution structural studies by NMR spectroscopy. After removal of the O-linked glycosylation sites by site-directed mutagenesis, we confirmed that the protein was no longer covalently glycosylated. However, analysis by 1H- and 13C-edited spectroscopy identified the presence of non-covalently associated glycans which were removed by lectin affinity chromatography. We have synthesised methods for the identification and removal of both covalently and non-covalently bound oligosaccharides from heterologous protein expressed in P. pastoris.
authors.affiliation: Division of Structural Biology, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. jo.oleary@bioch.ox.ac.uk
authors.forename: Joanne M
authors.initials: JM
authors.lastname: O'Leary
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Radcliffe
authors.forename: Antony C
authors.initials: AC
authors.lastname: Willis
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
authors.forename: A Kristina
authors.initials: AK
authors.lastname: Downing
mesh_terms: D002846:Chromatography, Affinity; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D037102:Lectins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009844:Oligosaccharides / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D011134:Polysaccharides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D037102:Lectins; D009844:Oligosaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins