Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 7 1 2015 05 22 Expression of recombinant gamma interferon in tobacco plant seeds Expression of recombinant gamma interferon in tobacco plant seeds 1 16 1348 10.22103/jab.2015.1348 FA Saber Asghar Zadeh Khadijeh Baqeri Mokhtar Jalali Javaran Fereydoun Mahboudi Journal Article 2013 03 09 Some important strategies to increase recombinant protein yield in plants include expression of protein in suitable tissue, improvement of protein stability and accumulation by targeting into subcellular organs using specific targeting signals. In this perspective, seed-based platforms are attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass, which is beneficial for extraction and downstream processing. On the other hand,  the ER contain chaperons and isomerase for folding of nascent proteins, which can promote correct protein folding leading to recombinant protein stability and accumulation. In this research for human Gamma interferon expression in tobacco seeds and targeting into ER, we used seed specific promoter (Napin), Kozak sequence at 5' end and KDEL sequence  in 3' end of the IFNγ gene.  The fragment was cloned into pGEM®-T Easy vector and after confirmation by PCR, restriction enzyme analysis and sequencing,  was subcloned into a plant binary vector (pBI121).  This construct cassette was transferred to A.tumefaciens LBA4404 and then used to transform tobacco explants.  The transformed plants were screened on antibiotic-contained media. The presence of the transgenes was confirmed in the transformants by PCR and expected 432bp (IFNγ) and 795bp (nptII) sequences were amplified.  Digestion result indicated that the mentioned construct completely and correctly transferred into tobacco genome. Transcription of  IFNγ gene was confirmed by RT-PCR. Analysis of transgenic plants by SDS-PAGE represented that IFNγ protein is being expressed in seeds. Our results indicate that plant seeds have potential for production of recombinant proteins as ‘natural bioreactors’ Some important strategies to increase recombinant protein yield in plants include expression of protein in suitable tissue, improvement of protein stability and accumulation by targeting into subcellular organs using specific targeting signals. In this perspective, seed-based platforms are attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass, which is beneficial for extraction and downstream processing. On the other hand,  the ER contain chaperons and isomerase for folding of nascent proteins, which can promote correct protein folding leading to recombinant protein stability and accumulation. In this research for human Gamma interferon expression in tobacco seeds and targeting into ER, we used seed specific promoter (Napin), Kozak sequence at 5' end and KDEL sequence  in 3' end of the IFNγ gene.  The fragment was cloned into pGEM®-T Easy vector and after confirmation by PCR, restriction enzyme analysis and sequencing,  was subcloned into a plant binary vector (pBI121).  This construct cassette was transferred to A.tumefaciens LBA4404 and then used to transform tobacco explants.  The transformed plants were screened on antibiotic-contained media. The presence of the transgenes was confirmed in the transformants by PCR and expected 432bp (IFNγ) and 795bp (nptII) sequences were amplified.  Digestion result indicated that the mentioned construct completely and correctly transferred into tobacco genome. Transcription of  IFNγ gene was confirmed by RT-PCR. Analysis of transgenic plants by SDS-PAGE represented that IFNγ protein is being expressed in seeds. Our results indicate that plant seeds have potential for production of recombinant proteins as ‘natural bioreactors’ https://jab.uk.ac.ir/article_1348_14f1209a5bdc934cf67eaa8da38c78a7.pdf