Indiscriminate glycosylation of procarboxypeptidase Y expressed in Pichia pastoris.
To obtain large amounts of deglycosylated procarboxypeptidase Y (proCPY), in which all of the N-glycosylation sites were replaced by alanine residue by the point mutation method, an expression system was constructed using Pichia pastoris. The secreted enzyme was characterized by SDS-PAGE, native PAGE, MALDI-TOF mass spectrometry, and dynamic light scattering, and the results indicated heterogeneity. The recombinant proCPY contained 29 mol of glucose per mole of protein in average, according to the carbohydrate analysis by the phenol-sulfuric acid method. A large part of the recombinant enzyme absorbed on a Con A column: even the break-through fraction of the column contained 3 mol of glucose per mole of protein. These carbohydrates were removed by the mild alkaline treatment. Since the entire N-glycosylation site had been destructed in the present expression system, the carbohydrates contained in the recombinant proCPY are concluded to be O-linked ones, which bound indiscriminately to serine and/or threonine residues.
Maeda
Haruko
H
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Chatani
Eri
E
Koyama
Takahiro
T
Sugiura
Masatoshi
M
Izumi
Hirohisa
H
Hayashi
Rikimaru
R
D000409:Alanine / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry; D043402:Cathepsin A / Q000096:biosynthesis* / Q000737:chemistry; D002942:Circular Dichroism; D003208:Concanavalin A / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000737:chemistry; D006031:Glycosylation; D008027:Light; D019800:Phenol / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011498:Protein Precursors / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002241:Carbohydrates; D011498:Protein Precursors; D011994:Recombinant Proteins; D003208:Concanavalin A; D019800:Phenol; D043402:Cathepsin A; D005947:Glucose; D000409:Alanine