Application of Anoxia Conditions to Enhance Production of hCTLA4Ig in Transgenic Rice Suspension Cell Cultures

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced inducibly by sugar starvation using RAmy3D promoter in transgenic rice cell suspension cultures. Although RAmy3D promoter is known to be the strongest promoter in plant systems, main obstacle is that the target protein was produced in sugar-free conditions. To improve the production of hCTLA4Ig, we applied anoxia conditions with 30 mM of glucose to the production phases.The anoxia conditions with 30 mM of glucose at the production phases markedly improved cell viability under anoxia conditions. A viability level of more than 65% could be maintained for up to 30 days. Furthermore, the repression of the RAmy3D promoter by residual sugar in the production of hCTLA4Ig was not observed under anoxia conditions with 30 mM of glucose.In addition, the production periods of hCTLA4Ig was expanded up to 30 days, and the maximum production levels of hCTLA4Ig were 29 mg/L under anoxia conditions and 14 mg/L in aerobic conditions, respectively. Under anoxia conditions with 30 mM of glucose, alcohol dehydrogenase II gene was found to be strongly expressed. Therefore, we suggest that anoxia conditions could be used to achieve energy source by ethanolic fermentation for the improvement of the productivity of hCTLA4Ig in transgenic rice suspension cell cultures.