抄録
The baculovirus-insect cell system has been shown to be an excellent expression system capable of providing large quantities of various biologically active recombinant proteins. With the aim of developing an efficient protein production process using this system, we here investigated the production of β-galactosidase by immobilized Sf9 cells after infection with recombinant Autographa californica nucleopolyhedrovirus (AcNPV). When non-immobilized cells were transferred from TNM-FH supplemented with 10% fetal bovine serum (FBS) into serum-free TNM-FH on post-infection day 1, the β-galactosidase yield was found to be nearly equal to that obtained in the continuous presence of FBS, suggesting that FBS was essential up to post-infection day 1. This finding offers a promising means of recombinant protein production: a two-step culture in which cells are first grown and virus-infected in a serum-supplemented medium and recombinant protein is then produced in a serum-free basal medium after medium replacement on post-infection day 1. In order to carry out medium replacement easily, Sf9 cells were passively immobilized in reticulated polyvinyl formal (PVF) resin biomass support particles (BSPs; 2 x 2 x 2 mm cubes; pore diameter, 30-50 µm). When TNM-FH supplemented with 10% FBS was replaced with serum-free TNM-FH on post-infection day 1, cells immobilized at a density over 107 cells/cm3-BSP gave a high -galactosidase yield comparable to that obtained in a culture using the serum-supplemented TNM-FH throughout. In this two-step culture, recombinant protein production by densely immobilized cells is achieved in protein-free basal media, which can facilitate downstream processing and purification of target proteins and thereby reduce production costs.
