Improvement of Endo-β-N-acetylglucosaminidase H production using silkworm-baculovirus protein expression system

Abstract 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.