Synthesis of Neoglycoenzymes with Homogeneous N-Linked Oligosaccharides Using Immobilized Endo-β-N-acetylglucosaminidase A

• Published in: Biochemical and biophysical research communications Vol. 267; no. 1; pp. 134 - 138
• Main Authors: Fujita, Kiyotaka, Tanaka, Naotaka, Sano, Mutsumi, Kato, Ikunoshin, Asada, Yasuhiko, Takegawa, Kaoru
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.01.2000
• Subjects: Animals; Arthrobacter protophormiae; Carbohydrate Sequence; Cattle; Chromatography, Affinity; Cloning, Molecular; endo-^b-N-acetylglucosaminidase A; Enzymes - chemical synthesis; Enzymes, Immobilized - isolation & purification; Enzymes, Immobilized - metabolism; Escherichia coli; Glutathione Transferase; Glycopeptides - chemical synthesis; Glycoproteins - chemical synthesis; Glycosylation; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - isolation & purification; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - metabolism; Molecular Sequence Data; oligosaccharides; Oligosaccharides - chemistry; Pancreas - enzymology; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Ribonucleases - isolation & purification; Ribonucleases - metabolism
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1999.1963

A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was overexpressed in Escherichia coli as a fusion protein linked to glutathione S-transferase (GST). GST–Endo-A fusion was extracted as a soluble protein. The fusion protein was purified to homogeneity with glutathione–Sepharose 4B and showed transglycosylation activity toward high-mannose-type glycopeptides without removing the GST moiety. The GST–Endo-A immobilized on glutathione–Sepharose 4B retained its transglycosylation activity. The immobilized enzyme could transfer (Man)6GlcNAc en bloc to partially deglycosylated ribonuclease B without damaging its enzyme activity. The immobilized GST–Endo-A should be very useful for synthesizing active neoglycoenzymes attached with homogeneous N-linked oligosaccharides.