Jong-Moon Cho

Effect of bacitracin on hGM-CSF production in suspension cultures of transgenic Nicotiana tabacum cells

Bacitracin, a known protease inhibitor, successfully delayed the rate of proteolytic degradation of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF). In transgenic Nicotiana tabacum suspension cell cultures, no growth inhibition was observed when at most 500 mg l -1 bacitracin was added. However, cell growth rate and cell size index were significantly decreased by 1000 mg l -1 bacitracin and the maximum cell concentration was reduced from 17.1 to 11.2 g l -1 . In terms of hGM-CSF production, all cultures treated with bacitracin showed improved hGM-CSF production, but the protective effect of bacitracin was diminished in relation to culture time and the decrement of its concentration. Considering both the cell growth and protein production, 500 mg l -1 bacitracin showed the best results and improved the hGM-CSF production to 112.5 μg l -1 , which was 2.18-fold higher than the 51.5 μg l -1 of control with no bacitracin. There was no growth retardation or sudden drop in hGM-CSF concentration. The hGM-CSF concentration was increased up to 127.9 μg l -1 with 1000 mg l -1 bacitracin, even though this severelv inhibited the cell growth.

Increased hGM-CSF production and secretion with Pluronic F-68 in transgenicNicotiana tabacum suspension cell cultures

The effects of Pluronic F-68, a nonionic surfactant, on the production and secretion of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in a transgenicNicotiana tabacum cell suspension culture were investigated in this study. The addition of Pluronic F-68 was shown to extend cell survival in the stationary phase, but had no influence on effective initial cell growth. With regard to production, it increased the level of extracellular hGM-CSF two-fold. This may be attributable not only to the enhanced expression level, but also to the improved permeability of the cell membrane due to the interaction between Pluronic F-68 and the cell membrane and cell wall. The effect of Pluronic F-68 on the production and secretion of hGM-CSF in a bioreactor was also evaluated. hGM-CSF production in the bioreactor after the addition of Pluronic F-68 proved more effective than in flask cultures.

Stability enhancement of hGM-CSF in transgenicNicotiana tabacum suspension cell cultures

Proteolytic enzymes existing in plant cell cultured media are the major reason for the loss of secreted human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The addition of 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 with chemical protease inhibitors. Gelatin, as a competitive substrate for protease, 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 significantly stimulated the hGM-CSF production and accumulation in culture media, with no growth inhibition. Compared to the controls (4.72 μg/L), the extracellular hGM-CSF level could be increased to 39.78 μg/L with the addition of 5 g/L of gelatin.