فهرست مطالب bagheri. kh

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 plant tissue and improvement of protein stability and accumulation by targeting into subcellular organs using specific targeting signals. The ER contain different molecular chaperons and isomerase for folding of nascent proteins، which can prevent aggregation and formation of incorrect three dimensional structure and promote correct protein folding leading to recombinant protein stability and accumulation. In this perspective، seed-based platforms are particularly 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. In this research for human Gamma interferon expression in Brassica napus seeds and targeting into ER، we used seed specific promoter (Napin) and C-terminal KDEL sequence (ER retention signal). The fragment was cloned into pGEM®-T Easy vector and then was confirmed by PCR، restriction enzyme analysis and sequencing. The authentic PCR fragment was subcloned into a plant binary vector (pBI121). This construct cassette (pBI IFNγ) was transferred to A. tumefaciens LBA4404 and then used to transform B. napus using an Agrobacterium-mediated petiole cotyledonary transformation. The transformed plants were screened on antibiotic-contained media The presence and expression of the transgene was confirmed in the transformants by PCR and SDS-PAGE. ELISA test was used for biological activity.

The oleosin fusion protein provide a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of desired polypeptide. By fuseing oleosin to desired gene and using seed specific promoter,targeting of recombinant protein to seed oil bodies will be possible. In this study, we used plant oleosin as a carrier for the production of the human gamma interferon protein in Brassica napus seeds. At the first step, oleosin gene was isolated from Brassica napus genome and cloned in pBI121 plant binary vector. Human gamma interferon gene was cloned downstream of oleosin and proteolytic cleavage site was set between them. The interferonoleosin fusion gene was under the control of seed specific promoter (Napin). Cotyledon explant and A. tumefaciens (LBA4404) were used for plant transformation. The transformed shoots were screened on kanamycin contained media. The presence and expression of the transgene was confirmed in the transformants by PCR and RT-PCR.