مجله بیوتکنولوژی کشاورزی (May 2016)
Enhanced expression of tissue plasminogen activator via gene silencing suppressor strategy using P19 protein
Abstract
Human tissue-type plasminogen activator (tPA) is one of the most important therapeutically proteins involved in the breakdown of blood clots of brain and heart blood vessels following stroke. Therefore, several modern approaches in plant biotechnology such as nuclear and chloroplast transformation have been used to produce the valuable protein. Plant transient expression is a rapid, flexible and reproducible approach to obtain high-level of expression of valuable recombinant pharmaceutical proteins. It is shown that post-transcriptional gene silencing (PTGS) process influences the expression level. Therefore, the effect of co-expression of gene silencing suppressor gene P19, derived from tomato bushy stunt virus (TBSV), has been studied on transient expression of tPA in Nicotiana tabacum cv. Xanthi. To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pTRAc-tPA-ERH with Agrobacterium containing pCambia-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pTRAc-tPA-ERH. ELISA results from co-agroinjection experiments have shown that the expression level of tPA using p19 was 10mg/g of leaf fresh weight, which this expression level was about two times more than that from leaves injected with Agrobacterium without the vector harboring p19. Therefore, comparing to the stable transformation which needs more time to produce the protein, in transient expression, depends on the number of the injectable leaves per plant and production of 10mg of tPA per gram leaf fresh weight, a higher level of expression can be achieved less than one week. In conclusion, it seems transient expression could be utilized to express tPA usefully and the expression level could ameliorate using virus coded gene silencing suppressor to achieve the expression proportion of 10mg/g of leaf fresh weight.
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