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Expression and large-scale production of the biochemically active human tissue-plasminogen activator in hairy roots of Oriental melon (Cucumis melo)

Author:
Kim, Sung-Ryong, Sim, Joon-Soo, Ajjappala, Hemavathi, Kim, Yong-Hwan, Hahn, Bum-Soo
Source:
Journal of bioscience and bioengineering 2012 v.113 no.1 pp. 106-111
ISSN:
1389-1723
Subject:
Cucumis melo, DNA, RNA, Rhizobium rhizogenes, Western blotting, blood vessels, enzyme-linked immunosorbent assay, fibrin, fibrinolysis, gene expression, genes, genetically modified organisms, humans, melons, pH, plasmin, plasminogen, reverse transcriptase polymerase chain reaction, roots
Abstract:
Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating plasminogen to plasmin in blood vessels. t-PA and synthetic t-PA (st-PA) genes were expressed as enzymatically active form in hairy roots of Oriental melon (Cucumis melo L. cv. Geumssaragi-euncheon) infected by Agrobacterium rhizogenes. The insertion of the t-PA genes in genomic DNA of transgenic hairy roots was verified by PCR. The presence and expression of t-PA-specific transcripts in the total RNAs of transgenic hairy roots were confirmed by RT-PCR. Western blot analysis of the transgenic hairy roots showed a single major band of 59-kDa recombinant t-PAs. ELISA demonstrated that the highest level of recombinant t-PA (798ngmg⁻¹) was detected in hairy roots expressing t-PA. Similarly, the maximum fibrinolysis of recombinant t-PAs was observed in hairy roots transformed with t-PA. WPM medium was found to be more suitable for rapid growth of hairy roots among all the seven media types tested. The hairy root production was 5.8 times higher than that of White medium. The total yield of hairy roots grown on WPM medium was 621.8±8.7gL⁻¹ at pH 7.0. These studies demonstrate that the hairy roots could be employed for the mass production of enzymatically active t-PA.
Agid:
1127616