(10a) Improved Expression of Recombinant Protein Using a Cysteine Protease and Chitinase Deficient Bombyx Mori Multiple Nucleopolyhedrovirus Bacmid System in Silkworm Larvae

Silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. We developed the first practical Bombyx mori multiple nucleopolyhedrovirus (BmMNPV) bacmid system directly applicable for the protein expression of silkworm. The bacmid system of Bombyx mori multiple nucleopolyhedrovirus (BmMNPV) with the cysteine protease (BmMNPV-CP^-), and both the cysteine protease and chitinase genes deleted (BmMNPV-CP^--Chi^-) was constructed using the lambda recombination system. The protease activities of Bombyx mori cells and silkworm larvae infected with this BmMNPV-CP^- bacmid were reduced by 94% and 85%, respectively. When silkworm was infected with BmMNPV-CP^--Chi^- the viral protease and chitinase activities in the hemolymph of silkworm larvae were reduced by 95% and 50%, respectively. By using these systems, a human β1,3-N-acetylglucosaminyltransferase 2 fused with green fluorescent protein (GFP_uv-β3GnT2) fusion protein was successfully expressed in silkworm larvae with less protein degradation and without larvae liquefaction; β3GnT2 activity improved by 2.8-fold to that of conventional bacmid. This BmMNPV-CP^- or BmMNPV-CP^--Chi^- bacmid system provides rapid protein production in silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation and thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.