Expression and large-scale production of human tissue plasminogen activator (t-PA) in transgenic tobacco plants using different signal peptides.
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.
Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. ghasemie.hojjat@gmail.com
Hojjat Ghasemi
HG
Goojani
Mokhtar Jalali
MJ
Javaran
Jaber
J
Nasiri
Esmaeel Ghasemi
EG
Goojani
Houshang
H
Alizadeh
D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D030821:Plants, Genetically Modified; D021382:Protein Sorting Signals; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*
D021382:Protein Sorting Signals; D010959:Tissue Plasminogen Activator