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Expression and Large-Scale Production of Human Tissue Plasminogen Activator (t-PA) in Transgenic Tobacco Plants Using Different Signal Peptides

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Abstract

An attempt was made to assess the expression level and targeting of a human protein entitled recombinant tissue plasminogen activator (rt-PA) through accumulation in three cellular compartments including the endoplasmic reticulum and cytosolic and apoplastic spaces in transgenic tobacco plants. In this context, three chimeric constructs pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA were employed and transferred into the tobacco plants through a popular transformation-based system called Agrobacterium tumefaciens. As an initial screening system, the incorporation of the rt-PA gene in the genomic DNA of tobacco transgenic plants and the possible existence of the rt-PA-specific transcript in the total RNAs of transgenic plant leaves were confirmed via PCR and reverse transcription (RT)-PCR, respectively. Southern blot analysis, in addition, was used to determine the copy number of the corresponding gene (i.e., t-PA) transformed into the each transgenic plant; one or more copies were detected regarding transformants derived from all three abovementioned constructs. According to the enzyme-linked immunosorbent assay, the mean values of t-PA expression were calculated as 0.50, 0.68, and 0.69 μg/mg of the total soluble protein when a collection containing 30 transgenic plants transformed with pBI-SP-tPA, pBI-tPA-KDEL, and pBI-Ext-tPA was taken into account, respectively. The zymography assay was lastly performed and concluded the expression of the properly folded rt-PA in this expression system. Our results, altogether, revealed that tobacco plants could be utilized as a bioreactor system for the large-scale production of enzymatically active t-PA and presumably other therapeutic recombinant proteins in large quantities.

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Authors and Affiliations

  1. Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

    Hojjat Ghasemi Goojani & Mokhtar Jalali Javaran

  2. Department of Agronomy and Plant Breeding, Division of Molecular Plant Genetics, University of Tehran, Karaj, Tehran, Iran

    Jaber Nasiri

  3. Department of Agronomy and Plant Breeding, Agricultural College, University of Tehran, Karaj, Iran

    Esmaeel Ghasemi Goojani

  4. Department of Agricultural Biotechnology, Agricultural College, University of Tehran, Karaj, Iran

    Houshang Alizadeh

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  1. Hojjat Ghasemi Goojani
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  2. Mokhtar Jalali Javaran
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  4. Esmaeel Ghasemi Goojani
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Correspondence to Houshang Alizadeh.

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Goojani, H.G., Javaran, M.J., Nasiri, J. et al. Expression and Large-Scale Production of Human Tissue Plasminogen Activator (t-PA) in Transgenic Tobacco Plants Using Different Signal Peptides. Appl Biochem Biotechnol 169, 1940–1951 (2013). https://doi.org/10.1007/s12010-013-0115-4

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