Makoto Ogata1, Makoto Nakajima1, Takeomi Murata2, Taichi Usui2, and Enoch Y. Park1. (1) Laboratory of Biotecnology, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8017, Japan, (2) Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8017, Japan
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).