title: N-linked glycan characterization of heterologous proteins.
abstract: Our laboratory has focused on the re-engineered of the secretory pathway of Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans and other higher mammals (1,2). A reporter protein with a single N-linked glycosylation site, a His-tagged Kringle 3 domain of human plasminogen (K3), was used to identify combinations of optimal leader/catalytic domain(s) to recreate human N-glycan processing in the Pichia system. In this chapter we describe detailed protocols for high-throughput purification of K3, enzymatic release of N-glycans, matrix-assisted laser desorption ionization time-of-flight and high-performance liquid chromatography analysis of the released N-glycans. The developed protocols can be adapted to the characterization of N-glycans from any purified protein expressed in P. pastoris.
authors.affiliation: GlycoFi Inc., Lebanon, NH, USA.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Robert G
authors.initials: RG
authors.lastname: Miele
authors.forename: Teresa I
authors.initials: TI
authors.lastname: Mitchell
authors.forename: Tillman U
authors.initials: TU
authors.lastname: Gerngross
mesh_terms: D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D018082:Kringles; D009844:Oligosaccharides / Q000032:analysis; D010843:Pichia; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009844:Oligosaccharides; D011134:Polysaccharides; D011506:Proteins; D006026:Glycoside Hydrolases