Enhanced Production of Human Granulocyte-Macrophage Colony-Stimulating Factor from Transgenic Nicotiana tabacum Suspension Cells

초록

Pluronic F-68 and Tween 80 were found reduced the cell surface hydrophobicity significantly by adsorbing on plant cell surface. On the contrary, PEG 8,000 showed the opposite result. Surface-active additives were known to be adsorbed to cell surface and affect plant cell physiology. In this study, effects of surfactants on transgenic plant cell growth and the production of foreign proteins were examined. When 5 g/L of Pluronic F-68 was added, the production of extracellular human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was increased 4.9-fold compared to control without any cell growth inhibition. Short term changes of hGM-CSF secretion from N. tabacum cells treated with Pluronic F-68 also supported the permeabilizing effect. Among the types of polyethylene glycol, PEG 3,350 gave the best result and PEG with higher molecular weight reduced the beneficial effect on hGM-CSF production. Tween 20 and dimethyl sufoxide stimulated the secretion of hGM-CSF in spite of noticeable reduction of cell growth. In addition, proteolytic enzymes secreted into plant cell cultured media are the major reason for the loss of secreted hGM-CSF. The addition of protease inhibitors such as pepstatin, aprotinin and PMSF relatively decreased the proteolytic degradation of hGM-CSF in a conditioned medium, but sufficient prevention against the proteolytic activity could not be obtained. Gelatin, as a competitive substrate for proteases, showed a stabilizing effect in a conditioned medium. Compared to the initial hGM-CSF concentration in a conditioned medium, with 10 g/L of gelatin, 68% of the hGM-CSF remained after 5 days. In a cell culture experiment, 5 g/L of gelatin stimulated the hGM-CSF production significantly and accumulation in culture media without growth inhibition. Compared to the control cultures (4.7 ?/L), the extracellular hGM-CSF level could be increased to 39.8 ?/L with the addition of 5 g/L of gelatin.