Expression of recombinant rabbit UDP-GlcNAc: alpha 3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I catalytic domain in Sf9 insect cells.
UDP-GlcNAc: alpha 3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyses a key reaction in the conversion of oligomannose to complex and hybrid N-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 of Autographa 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 l-1) in two steps to a specific activity of about 2 mumol min-1 mg-1 protein. Recombinant GnT I has been used for the chemical-enzymatic synthesis of analogues of Man alpha 1-6]GlcNAc beta 1-2Man alpha 1-3]Man beta-O-octyl.
Sarkar
M
M
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
D000818:Animals; D001665:Binding Sites; D015139:Blotting, Southern; D002240:Carbohydrate Sequence; D002384:Catalysis; D002478:Cells, Cultured; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I