title: An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris.
abstract: The receptor for the advanced glycation end products (RAGE) is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and binds a variety of unrelated ligands sharing a negative charge. Most ligands bind to the extracellular V or VC1 domains of the receptor. In this work, V and VC1 of human RAGE were produced in the methylotrophic yeast Pichia pastoris and directed to the secretory pathway. Fusions to a removable C-terminal His-tag evidenced proteolytic processing of the tag by extracellular proteases and also intracellular degradation of the N-terminal portion of V-His. Expression of untagged forms was attempted. While the V domain was retained intracellularly, VC1 was secreted into the medium and was functionally active in binding AGEs. The glycosylation state of VC1 was analyzed by mass spectrometry and peptide-N-glycosidase F digestion. Like RAGE isolated from mammalian sources, the degree of occupancy of the N-glycosylation sites was full at Asn25 and partial at Asn81 which was also subjected to non-enzymatic deamidation. A simple procedure for the purification to homogeneity of VC1 from the medium was developed. The folded state of the purified protein was assessed by thermal shift assays. Recombinant VC1 from P. pastoris showed a remarkably high thermal stability as compared to the protein expressed in bacteria. Our in vivo approach indicates that the V and C1 domains constitute a single folding unit. The stability and solubility of the yeast-secreted VC1 may be beneficial for future in vitro studies aimed to identify new ligands or inhibitors of RAGE.
authors.affiliation: Department of Biosciences, Via Celoria 26, University of Milan, 20133 Milano, Italy.
authors.forename: Genny
authors.initials: G
authors.lastname: Degani
authors.affiliation: Department of Pharmaceutical Sciences, Via Mangiagalli 25, University of Milan, 20133 Milano, Italy.
authors.forename: Mara
authors.initials: M
authors.lastname: Colzani
authors.affiliation: Department of Biosciences, Via Celoria 26, University of Milan, 20133 Milano, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Tettamanzi
authors.affiliation: Department of Biosciences, Via Celoria 26, University of Milan, 20133 Milano, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Sorrentino
authors.affiliation: Department of Biosciences, Via Celoria 26, University of Milan, 20133 Milano, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Aliverti
authors.affiliation: Institute for Neuropathology, University of Freiburg, Breisacher Str. 64, 79106 Freiburg, Germany.
authors.forename: Guenter
authors.initials: G
authors.lastname: Fritz
authors.affiliation: Department of Pharmaceutical Sciences, Via Mangiagalli 25, University of Milan, 20133 Milano, Italy.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Aldini
authors.affiliation: Department of Biosciences, Via Celoria 26, University of Milan, 20133 Milano, Italy. Electronic address: Laura.Popolo@unimi.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Popolo
mesh_terms: D006031:Glycosylation; D010843:Pichia / Q000235:genetics*; D055550:Protein Stability; D017434:Protein Structure, Tertiary / Q000235:genetics; D000067759:Receptor for Advanced Glycation End Products / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000067759:Receptor for Advanced Glycation End Products; D011994:Recombinant Proteins
keywords: Deamidation; Immunoglobulin superfamily; Pichia pastoris; Protein N-glycosylation; RAGE; Receptors