Development of silkworm virus-derived, multi-layered microcrystals encapsulating two different proteins in the different layers

抄録

Many proteins have been shown to be encapsulated in cypovirus (BmCPV) protein microcrystal polyhedra. However, a method to control the BmCPV polyhedron portion for encapsulation has not been elucidated. Herein, we report the development of a method to produce multi-layered BmCPV polyhedra. For this, isolated cultured Sf21 cells that stably expressed BmCPV polyhedra encapsulation signal (H1)-fused EGFP (H1-EGFP) were obtained, in which baculovirus carrying both BmCPV polyhedrin and H1-DsRed produced high percentage (91.56%) of multi-layered BmCPV polyhedra with H1-EGFP encapsulated in the inner layer and H1-DsRed in the outermost. In addition, we generated transgenic silkworms with cells, in which the H1-EGFP was continuously expressed and the recombinant baculovirus produced 82.33% of multi-layered BmCPV polyhedra. These results indicate that foreign proteins already abundant in the cells before the start of BmCPV poryhedrin crystallization are encapsulated in the inner layer of the polyhedra, whereas foreign proteins expressed simultaneously with BmCPV polyhedrin were encapsulated in the outermost layer. Dissolving the multi-layered polyhedra in buffer (pH 9.5) revealed the first phase of the outermost H1-DsRed release, followed by the second phase of inner H1-EGFP release, demonstrating that the multi-layered BmCPV polyhedra can release encapsulated proteins with different timings, which can be applied to the development of drug delivery carriers applicable for tissue engineering.