Adsorption of secreted protease by using silica gel to reduce protein degradation in transgenic rice cell suspension cultures

초록

Recently, transgenic plant cells have been used as a host system for the production of therapeutic proteins, categorized as plant-made pharmaceuticals (PMPs). Transgenic rice cells were used to produce human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) which is a fusion protein used for the treatment of rheumatoid arthritis. By using RAmy3D promoter, hCTLA4Ig was induced strongly by sugar starvation. However, the sugar-starved environment causes cell death during production phase. Cell death also affects the protein degradation due to secreted proteases by cell lysis. Mesoporous silica materials are a class of inorganic materials with large surface area, well-ordered porous structures, and reproducible pore-size distributions with relatively large-pore diameters (2–40 nm). Due to these characteristics, the porous silica materials have been known as immobilizing materials for the diverse sizes of proteins. Therefore, various enzymes could be bind to the surfaces of silica materials via various forces, such as electrostatic interactions, hydrogen bonding, and van der Waals interactions. In recent investigation, commercial silica gel was found to be strongly interacted with trypsin. In this study, the adsorption of proteases by using silica gel was investigated for reducing protein degradation of produced hCTLA4Ig in transgenic rice cell suspension cultures. Analyses of protease activity, total protein, and hCTLA4Ig were performed to identify the adsorption of protease by silica gel. To optimize the concentration of silica gel, various concentrations of silica gel were added to 100-mL Erlenmeyer flasks. Addition time of silica gel to the culture media was also investigated to reduce protease activity. In conclusion, protein degradation could be lowered by the adsorption of proteases to silica-gel in transgenic rice cell suspension cultures.