Expression of Tissue-Type Plasminogen Activator and Its Derivative Proteins in Transgenic Tobacco Plants
2009
Kim, H.M. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea) | Sim, J.S. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea) | Rhee, Y. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea) | Pak, H.K. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea) | Yoon, M.S. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea) | Lim, K.H. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea) | An, K.S. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea) | Kim, Y.H. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea) | Hahn, B.S. (National Academy of Agricultural Science, RDA, Suwon, Republic of Korea), E-mail: bshahn@rda.go.kr
t-PA is a proteolytic enzyme found in blood and tissues. The major physiological function of t-PA is to generate plasmin that can dissolve blood clots in the blood vessel. To express recombinant t-PA derivative proteins (t-PAER and t-PAHis∧6) and t-PA protein encoded by synthetic t-PA gene in plants, the DNA fragments encoding the synthetic t-PA protein encoded by synthetic t-PA gene in plants, the DNA fragments encoding the synthetic t-PA and t-PA derivative proteins were cloned into a plant binary plasmid that included the cauliflower mosaic virus 35S promoter, a tobacco etch virus (TEV) leader sequence, an N-terminal signal peptide of the alfala glucose-regulated endoplasmic recticular protein and a 35S terminator. The insertion of the synthetic t-PA, t-PAER and t-PAHis∧6 genes in genomic DNA of tobacco plants was confirmed by PCR. Western blot analysis of the transformed tobacco leaves revealed single bands of approximately 68-kDa recombinant t-PA and its derivative proteins. ELISA experiments demonstrated that the highest level of recombinant t-PA expression was 0.0978 ㎍/total soluble protein (mg) in tobacco plants. The amount of recombinant t-PA and its derivative proteins in transgenic plants was estimated to range from 0.0011 to 0.0098% of total soluble proteins. The fibrinolysis of recombinant t-PA and its derivative (t-PAER and t-PAHis∧6) proteins was confirmed by a fibrin plate assay. These studies demonstrated that plants could be employed as bioreactor systems for lower cost t-PA production and may aid basic study for the high production of humanized t-PA.
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