Expression and Establishment of the Optimum Conditions for the Mass-Production of the Biochemically Active Human Tissue-Plasminogen Activator in Hairy Roots of Cucumis Melo (Oriental Melon)

Human tissue-plasminogen activator (t-PA), its derivatives and synthetic genes were expressed as enzymatically active form from hairy roots of Cucumis melo L. cv. Geumssaragi-euncheon (Oriental melon) infected by Agrobacterium Rhizogenes strains K599 harboring binary vectors. The insertion of the t-PA genes in genomic DNA of transgenic hairy roots was verified by PCR. The presence of the t-PA-specific transcripts in the total RNAs of transgenic hairy roots was confirmed by RT-PCR. Western blot analysis of the transgenic hairy roots showed a single major band of 68-kDa recombinant t-PAs. ELISA experiments demonstrated that the highest level of recombinant t-PA expression was average 0.03% of the total soluble protein in hairy roots transformed by plasmid p221t-PAer.

The optimum condition for the growth of transgenic hairy roots were MS and WPM of optimal medium, 7.0 of pH level, sucrose of carbon source and 1% of sucrose concentration. After 14 days of inoculation the yield of hairy roots grown on MS medium(pH7.0) was 18 times higher than that of the immediately inoculation. The yield of hairy roots grown on MS medium was 618g at pH 7.0. These studies demonstrated that hairy roots could be employed for the mass production of an enzymatically active t-PA.

The project was supported by the grant from NAAS(200901FHT01047539)