Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-beta-N-acetylglucosaminidase A.
A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-beta-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)(6)GlcNAc 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.
Fujita
K
K
Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan.
Tanaka
N
N
Sano
M
M
Kato
I
I
Asada
Y
Y
Takegawa
K
K
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004798:Enzymes / Q000138:chemical synthesis*; D004800:Enzymes, Immobilized / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D005982:Glutathione Transferase; D006020:Glycopeptides / Q000138:chemical synthesis; D006023:Glycoproteins / Q000138:chemical synthesis*; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010179:Pancreas / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*
D004798:Enzymes; D004800:Enzymes, Immobilized; D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D012260:Ribonucleases; C029600:ribonuclease B; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase