No abstract

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.

No abstract

Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension culture medium, and manifested biological activity.

Dengue virus enters into host cells by binding envelope glycoprotein (E) to a receptor and causes human disease. Domain III of dengue virus E glycoprotein (EIII) is immunogenic and capable of inducing neutralizing antibodies. A DNA fragment encoding EIII (amino acids 297-394) was introduced into tobacco plant cells (káÅçíá~å~=í~Ä~Åìã L. cv MD609) by ^ÖêçÄ~ÅíÉêáìã=íìãÉÑ~ÅáÉåë-mediated transformation methods. The integration of EIII gene was observed in the genomic DNA of transgenic plants by PCR DNA amplification methods. The transcripts of EIII gene were detected in the transgenic plant leaf tissues by Northern blot analysis. The EIII protein was detected in transgenic plant leaf extracts by Western blot analysis. Based on the results of the Western blot analysis, the expression level of plantproduced EIII protein was between 0.13 and 0.25% of the total soluble protein in transgenic plant leaf tissues. The production of domain III of dengue virus E glycoprotein (EIII) in transgenic plants demonstrates the feasibility of using plant-based vaccines to prevent infection by the dengue virus. © KSBB hÉóïçêÇëW=ÇÉåÖìÉ=îáêìëI=éä~åíJÄ~ëÉÇ=î~ÅÅáåÉI=ãìÅçë~ä=áããìåáò~íáçåI=Ççã~áå=fff=çÑ=ÇÉåÖìÉ=îáêìë=ÉåîÉäçéÉ=ÖäóÅçéêçíÉáå= = = = =