Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-b-N-acetylglucosaminidase A

Kiyotaka Fujita, Naotaka Tanaka, Mutsumi Sano, Ikunoshin Kato, Yasuhiko Asada, Kaoru Takegawa

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

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. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)134-138
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume267
Issue number1
DOIs
Publication statusPublished - Jan 7 2000
Externally publishedYes

Fingerprint

Acetylglucosaminidase
Glutathione Transferase
Oligosaccharides
Fusion reactions
Sepharose
Glutathione
Arthrobacter
Immobilized Enzymes
Proteins
Gene encoding
Glycopeptides
Enzyme activity
Mannose
Escherichia coli
endo-alpha-sialidase
Enzymes
Genes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-b-N-acetylglucosaminidase A. / Fujita, Kiyotaka; Tanaka, Naotaka; Sano, Mutsumi; Kato, Ikunoshin; Asada, Yasuhiko; Takegawa, Kaoru.

In: Biochemical and Biophysical Research Communications, Vol. 267, No. 1, 07.01.2000, p. 134-138.

Research output: Contribution to journalArticle

Fujita, Kiyotaka ; Tanaka, Naotaka ; Sano, Mutsumi ; Kato, Ikunoshin ; Asada, Yasuhiko ; Takegawa, Kaoru. / Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-b-N-acetylglucosaminidase A. In: Biochemical and Biophysical Research Communications. 2000 ; Vol. 267, No. 1. pp. 134-138.
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