Expression and Characterization of Human Tissue-Plasminogen Activator in Transgenic Tobacco Plants

Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating the transition from plasminogen to plasmin in blood vessels. The recombinant tissue-plasminogen activator (rTPA) produced in plants is a soluble protein derived from human t-PA. In order to produce rTPA protein in plants, a DNA fragment encoding the t-PA protein was 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 reticular protein, and an 35S terminator. The insertion of the rTPA gene in genomic DNA of phospinothricin-resistant tobacco plants was confirmed by polymerase chain reaction (PCR). The presence of the rTPA-specific transcript in the total RNAs of transgenic plants leaves was verified by reverse transcription (RT)-PCR. Immunoblot analysis of the transformed tobacco leaves revealed single bands of 68-kDa rTPAs. Enzyme-linked immunosorbent assay experiments demonstrated that the highest level of rTPA expression was 0.0014% of the total soluble protein in tobacco plants. Fibrinolysis of rTPA was confirmed by fibrin plate assay and zymography. These studies demonstrate that plants can be employed as bioreactor systems for the production of an enzymatically active t-PA with properties similar to the recombinant t-PA produced by mammalian cells.