Glycoconjugate Journal

, Volume 11, Issue 3, pp 204–209 | Cite as

Expression of recombinant rabbit UDP-GlcNAc:α3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I catalytic domain in Sf9 insect cells

  • Mohan Sarkar
Article

Abstract

UDP-GlcNAc:α3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyses a key reaction in the conversion of oligomannose to complex and hybridN-glycans. The cytoplasmic tail and transmembrane segment of rabbit GnT I cDNA were replaced with an in-frame cleavable signal sequence and the hybrid construct was inserted into the genome ofAutographa californica nuclear polyhedrosis virus (AcMNPV) under the control of the polyhedrin promoter. Sf9 insect cells were infected with the recombinant baculovirus and the enzymatically active and soluble catalytic domain of GnT I was purified from the medium (1–5 mg 1−1) in two steps to a specific activity of abut 2 µmol min−1 mg−1 protein. Recombinant GnT I has been used for the chemical-enzymatic synthesis of analogues of Manα1-6[GlcNAcβ1-2Manα1-3]Manβ-O-octyl.

Keywords

recombinant N-acetylglucosaminyl transferaseI N-glycans baculovirus insect cells 

Abbreviations

AcMNPV

Autographa californica nuclear polyhedrosis virus

FCS

fetal calf serum

1 µmol min−1

international enzyme unit

MAG

myelin associated glycoprotein

MOI

multiplicity of infection

pfu

plaque forming units

SDS-PAGE

sodium dodecyl sulfate/polyacrylamide gel electrophoresis

Sf9 cells

Spodoptera frugiperda insect cells

GnT I

UDP-GlcNAc:α3-d-mannoside β1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)

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Copyright information

© Chapman & Hall 1994

Authors and Affiliations

  • Mohan Sarkar
    • 1
    • 2
  1. 1.Research Institute, The Hospital for Sick ChildrenTorontoCanada
  2. 2.The Protein Engineering Network of Centres of Excellence, Canada (PENCE)Canada

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