Transiently Expressed Human Tissue Specific Plasminogen Activator (t-PA) in Tobacco Leaves Dissolves Fibrin and Blood Clots

Transient expression in tobacco leaves has been a very effective method of recombinant protein production. In this study we used the germinivirus-based single DNA replicon system viral vector, pBYr2fp, to transiently express t-PA in tobacco leaves. The ER retention signal, KDEL, was used for foreign protein accumulation. The t-PA protein was localized in the apoplast with LPH signal (plant codon-optimized leader peptide (LPH) derived from the heavy chain of the murine mAb24). Agroinfiltration was used to transiently expressed t-PA in wild type tobacco leaves. Leaf necrosis was observed 3 days after infiltration. The protein was purified with His-Tag affinity chromatography and the protein size was confirmed using SDS-PAGE. It degraded artificial fibrin with fibrin plate assay. Blood clot lysis assay was also performed. Fibrin degradation product (FDP) and D-dimer assay were used to quantify fibrin degradation, respectively. Our study indicated that plant system can produce functional recombinant t-PA production.