earticle

영어

Recombinant protein production in plant cell suspension culture is limited by secreted proteases with high level. We identified secreted protease during rice cell suspension culture using two-dimensional electrophoresis (2-DE) and ESI/Q-TOF mass spectrometry analysis. Total secreted proteins were extracted from rice suspension cell culture at 6 days after sugar starvation by using phenol method. Thirty seven protein spots were induced or increased by the sugar starvation, which we analyzed by internal amino acid sequencing with ESI/Q-TOF mass spectrometry analysis. The internal amino acid sequences of five spots correspond to cysteine proteinase (CysP), encoded by Rep1. This result indicates that CysP is one of secreted protease during rice cell suspension culture. Intron-containing self complementary hairpin RNA (ihpRNA)-mediated post transcriptional gene silencing (PTGS) was applied to suppress the expression of CysP in rice cell suspension culture. Analysis of secreted total protease and CysP activity in the supernatant of transgenic rice cell suspension culture showed lower activity than those of wild type and mRNA level of a rice CysP gene in Northern blot analysis was also reduced remarkably in the transgenic rice callus. At the same time, the detection of 21-23nt siRNA, initiator of RNAi by RNase protection assay indicated that PTGS worked successfully in this system. It was investigated whether the production of hGM-CSF in rice cell line (pMYN44-08) which stably produced hGM-CSF could be improved by suppressing expression of CysP. The suspension culture of rice cell transformed with both the hGM-CSF and a dsRNA of CysP contained reduced total protease and CysP activities, and increased hGM-CSF production levels compared those of cell lines harboring the hGM-CSF only. The production of hGM-CSF increased by up to 2-5 folds at 5 days using RNAi technology described above. This study demonstrated that the expression of CysP, secreted protease in rice cell suspension culture was suppressed by siRNA, and degradation of hGM-CSF was protected from protease attacks.