Amylase and cysteine proteinase gene knockdown in rice cells using RNA interference for enhancing production of recombinant proteins

• Published in: Plant cell, tissue and organ culture tissue and organ culture, 2013-07, Vol.114 (1), p.97-107
• Main Authors: Kim, Nan-Sun, Jang, Seon-Hui, Yu, Hwa-Young, Chung, Nguyen-Duc, Kwon, Tae-Ho, Yang, Moon-Sik, Kim, Tae-Geum
• Format: Article
• Language: English
• Subjects: Accumulation; Amylases; Antibodies; Biomedical and Life Sciences; Biotechnology; Cell culture; Colony-stimulating factor; Cysteine; Cysteine proteinase; Depletion; Electrophoresis; Gel electrophoresis; Gene expression; Granulocyte-macrophage colony-stimulating factor; Interference; Life Sciences; Original Paper; Oryza; Oryza sativa; Plant Genetics and Genomics; Plant Pathology; Plant Physiology; Plant Sciences; Polymerase chain reaction; Protease; Proteinase; Proteins; Reduction; Reverse transcription; Ribonucleic acid; Rice; RNA; RNA-mediated interference; Sodium dodecyl sulfate; Sodium lauryl sulfate; Sucrose; Sugar; Suspension culture; α-Amylase
• ISSN: 0167-6857; 1573-5044
• DOI: 10.1007/s11240-013-0309-z

A rice cell suspension culture with the rice α-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 α-amylase and proteases in the culture medium. Rice α-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 α-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 α-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 α-amylase and CysP genes evidenced a reduction of α-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 α-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.