科研之友
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摘要
A rice cell suspension culture with the rice alpha-amylase 3D promoter expression system which is induced by sucrose starvation was previously reported to generate a good yield of recombinant proteins. However, this expression system is limited by the accumulation of undesirable alpha-amylase and proteases in the culture medium. Rice alpha-amylase is a dominant protein at 43 % of total secreted proteins, and cysteine proteinase (CysP) is a major secreted protease in rice cell suspension cultures following induction via sugar depletion. Here, we nearly eliminated rice alpha-amylase and CysP proteinase via RNA interference (RNAi) technology to improve the recombinant protein yield in rice cell suspension culture. The effects of RNAi were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis with anti-CysP antibody, and quantitative real-time reverse transcription polymerase chain reaction analysis (RT-PCR). The mRNA levels of alpha-amylase and CysP were reduced by 94.8 and 95.0 %, respectively. Transgenic rice cell suspension cultures expressing both human granulocyte-macrophage colony-stimulating factor (hGM-CSF) and ihpRNA of alpha-amylase and CysP genes evidenced a reduction of alpha-amylase and CysP activity and up to 2.4-fold improvement of hGM-CSF production compared to that in a transgenic cell line expressing hGM-CSF only. Our rice cell suspension culture for the reduction of alpha-amylase accumulation and protease activity in the culture medium could improve recombinant protein production as an efficient protein expression system using RNA interference technology in plant biotechnology.
- 出版日期2013-7
