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.
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.
García
R
R
GlycoLab, Bio Industry Division, Havana, Cuba.
Rodríguez
R
R
Montesino
R
R
Besada
V
V
González
J
J
Cremata
J A
JA
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*
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