title: Concanavalin A- and wheat germ agglutinin-conjugated lectins as a tool for the identification of multiple N-glycosylation sites in heterologous protein expressed in yeast.
abstract: We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase. Wheat germ agglutinin-horseradish peroxidase is used as the recognition system for peptides generated after proteolytic digestion of endoglycosidase H deglycosylated protein. Glycosylation sites were confirmed by automatic Edman degradation and fast atom bombardment mass spectrometry. This methodology was applied to a model glycoprotein, alpha-amylase from Bacillus licheniformis, which is unglycosylated in its natural host and appears highly glycosylated when expressed in the methylotrophic yeast Pichia pastoris.
authors.affiliation: GlycoLab, Bio Industry Division, Havana, Cuba.
authors.forename: R
authors.initials: R
authors.lastname: García
authors.forename: R
authors.initials: R
authors.lastname: Rodríguez
authors.forename: R
authors.initials: R
authors.lastname: Montesino
authors.forename: V
authors.initials: V
authors.lastname: Besada
authors.forename: J
authors.initials: J
authors.lastname: González
authors.forename: J A
authors.initials: JA
authors.lastname: Cremata
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus; D002240:Carbohydrate Sequence; D003208:Concanavalin A; D006031:Glycosylation; D006596:Hexosaminidases; D006735:Horseradish Peroxidase; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; C045779:concanavalin A-horseradish peroxidase conjugate; D003208:Concanavalin A; D006735:Horseradish Peroxidase; D006596:Hexosaminidases; D000516:alpha-Amylases