Expression And Assay Of Human β-1,2-N-acetylglucosaminyltransferase Ⅰ In Pichia Pastoris

Abstract: Pichia pastoris expression system is one of the most frequently used expression systems for producing foreign proteins. This study is indispensable to the yeast N-glycosylation engineering project in our lab. We planned to introduce a human β-1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) into the yeast genome, which catalyzes an essential first step in the conversion of high-mannose N-glycans to hybrid and complex N-glycans.The 1224bp gntl sequence was amplified by PCR from human blood genome DNA. The sequence encoding human GnTI Golgi location was replaced by the sequence which encodes S.cerevisise MNN9 Golgi-retaining location signal. And the myc-sequence was added to the end of the gntl and the chimeric DNA fragment was cloned into the expression vector of pAO815, resulting in pAO815-mnn9-gntl-myc (pAgm for short). Later, Pichia pastoris GS115 was transformed with plasmid pAgm. The transformants, GS115-pAgm was identified by genome PCR, RT-PCR and ELISA. Finally, successful in vitro transfer of GlcNAc was demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE), showing N-acetylglucosaminyltransferase activity in the genetic engineered Pichia pastoris.