Endo-β-. N-acetylglucosaminidase H (Endo H) catalyzes cleavage between the GlcNAc residues of the chitobiose core of N-linked glycans, leaving one GlcNAc residues attached to asparagine. Endo H cleaves high mannose and hybrid, but not complex, N-linked oligosaccharides on glycoproteins. Because of its unique specificity, Endo H is widely used for the structural and functional analyses of glycoproteins. In our previous study, the recombinant Endo H was produced as a secreted protein using silkworm-baculovirus expression system, but the yield was low (30. μg Endo H/10. ml larval hemolymph) compared to that of Escherichia coli. In this study, we purified active recombinant Endo H as an intracellular protein from fat body of silkworm infected with the recombinant baculovirus expressing Endo H without the exogenous signal peptide. Remarkably, the yield (9.3. mg from 20 silkworm larvae) was about 310-fold higher than that secreted into larval hemolymph as reported previously. In addition, we screened the silkworm strains maintained in Kyushu University and identified n17 as a high-level expression strain for Endo H.
All Science Journal Classification (ASJC) codes
- Insect Science