Recombinant protein production in plant cell suspension culture is limited by secreted proteases with high level. We used two-dimensional electrophoresis (2-DE) and proteomic approaches to identify protease expressed in rice suspension cell culture in ...

Recombinant protein production in plant cell suspension culture is limited by secreted proteases with high level. We used two-dimensional electrophoresis (2-DE) and proteomic approaches to identify protease expressed in rice suspension cell culture in sugar starvation condition. 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 Hybrid tandem MS (QSTAR). The internal amino acid sequence of eight spots correspond to cysteine endopeptidase (CysP), encoded by Rep1 that induced by sugar starvation in rice suspension cell culture. Rice chitinase class I precusor, putative receptor-like protein kinase, ribonuclease, ankyrin repeat protein family-like protein, putative beta-1,3 glucanase and putative phytocyanin-related protein were among those induced by sugar starvation in rice cell suspension culture. This result indicates that cysteine endopeptidase is major secreted protease during rice cell suspension culture.
RNA interference (RNAi) was utilized to examine the effects of suppressed 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 that of wild type. In the results of 2-D PAGE, the accumulation of rice CysP in rice cell suspension culture was significantly reduced compare to that of wild type. mRNA level of a rice CysP gene in Northern blot analysis was also reduced remarkably in the transgenic rice callus. And we detected 21-23nt siRNA , initiator of RNAi by RNase protection assay. Here, we have investigated whether the production in hGM-CSF could be improved by the simultaneous introduction of an dsRNA of CysP. The suspension culture of transformed rice cell with both the hGM-CSF and a dsRNA of CysP contained reduced total protease and CysP activity and increased hGM-CSF production levels compared to lines harboring the hGM-CSF only. We increased the production of hGM-CSF by up to 5-fold by using RNAi, indicating that it should that are intrinsically deficient in protease.

식물세포배양을 이용하여 의료용 단백질인 hGM-CSF의 생산성을 향상시키기 위해 최신 분자생물학적 기법을 응용한 논문으로 그 학문적 수준이 매우 높은 것으로 사료되며,아울러 재조합 단백질의 생산량이 250mg/L 로 산업화를 위한 가능성을 제시한다.

식물세포배양을 이용하여 의료용 단백질인 hGM-CSF의 생산성을 향상시키기 위해 최신 분자생물학적 기법을 응용한 논문으로 그 학문적 수준이 매우 높은 것으로 사료되며,아울러 재조합 단백질의 생산량이 250mg/L 로 산업화를 위한 가능성을 제시한다.