Efficient Expression and Purification of a2,6-Sialyltransferase in the Hemolymph of Bombyx Mori (silkworm) Larvae Using Bmnpv Bacmid and Its Application on Glycotechnology

In previous study we demonstrated, Bombyx mori nuclear polyhedrosis virus (BmNPV) bacmid system provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins (T. Motohashi et al., Biochem. Biophis. Res. Commun., 326 (1), 2005, 564-569). In this study, we performed expression and purification of a2,6-sialyltransferase (a2,6-SiaT) using B. mori and also its application on the synthesis of human type influenza virus infection inhibitor.

First, fusion gene encoding rat a2,6-SiaT containing bombyxin (bx) signal peptide, histidine (His) tag and enterokinase recognition site was constructed, and then we created BmNPV bacmid containing bx-His-a2,6-SiaT fusion gene. Moreover using bacmid system, the a2,6-SiaT was expressed in the hemolymph of silkworm larvae. a2,6-SiaT activity was 0.93 U/ml, and possessed strong sialic acid transfer activity. In addition, produced a2,6-SiaT was partial purified about 100-fold to homogeneity from crude extract of the B. mori silkworm larvae with a yield of 30% using a cation exchange chromatography.

This enzyme was shown to be useful for a service tool in the synthesis of human type influenza virus infection inhibitors (M. Ogata et al., Bioorg. Med. Chem., 15, 2007, 1383-1393).