Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture.
Recombinant proteins have been previously synthesized in a transgenic rice cell suspension culture system with the rice amylase 3D promoter, which can be induced via sugar starvation. However, the secreted recombinant proteins have been shown to be rapidly decreased as the result of proteolytic degradation occurring during prolonged incubation. The secreted proteases were identified via two-dimensional electrophoresis (2-DE) and ESI/Q-TOF mass spectrometry analyses. The internal amino acid sequences of 8 of 37 spots corresponded to cysteine proteinase (CysP), which is encoded for by Rep1 and EP3A. This result shows that CysP is a major secreted protease in rice cell suspension cultures following induction via sugar starvation. 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 cultures. The reduction of rice CysP mRNA and the detection of siRNA specific to CysP, an initiator of RNAi, were verified via Northern blot analysis and RNase protection assays, respectively, thereby indicating that PTGS operated successfully in this system. The analysis of total secreted protease and CysP activities evidenced lower activity than was observed with the wild-type. Furthermore, suspension cultures of rice cells transformed with both hGM-CSF and the gene expressing the ihpRNA of CysP evidenced a reduction in total protease and CysP activities, and an up to 1.9-fold improvement in hGM-CSF production as compared to that observed in a rice cell line expressing hGM-CSF only. These results demonstrate the feasibility of the suppression of CysP via RNA interference to reduce protease activity and to increase target protein accumulation in rice cell suspension cultures.
Kim
Nan-Sun
NS
Division of Biological Sciences, Chonbuk National University, Dukjindong 664-14, Jeonju, Chonbuk 561-756, Republic of Korea.
Kim
Tae-Geum
TG
Kim
Ok-Hyun
OH
Ko
Eun-Mi
EM
Jang
Yong-Suk
YS
Jung
Eun-Sun
ES
Kwon
Tae-Ho
TH
Yang
Moon-Sik
MS
D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins
D012333:RNA, Messenger; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003546:Cysteine Endopeptidases