Construction of A Screening System for Endo-β-N-Acetylglucosaminidase Using Chinese Hamster Ovary Cell

Abstract:

Endo-β-N-Acetylglucosaminidase belonging to glycoside hydrolase family 85（ENGase） exhibits transglycosylation activity，which is a useful tool for the synthesis of glycoconjugates with a defied structure of N-glycan. In the present study，We constructed a cell-based screening system to obtain mutant ENGase. As a substrate for ENGase，we expressed monomer enhanced green fluorescent protein（mEGFP） with N-glycan in the Golgi of Chinese hamster ovary cells. Cells expressing N-glycosylated mEGFP provided 10 times lower fluorescence than those expressing mEGFP. Inhibition of protein glycosylation by tunicamycin caused further decrease of fluorescence from the N-glycosylated mEGFP. Our results suggest that this cell line can be used for the high throughput screening of mutant ENGase.