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To explore a cost-effective alternative method to produce the recombinant thrombolytic drug Reteplase (rPA), a plant viral amplicon-based gene expression system was employed to transiently express bioactive Strep II-tagged recombinant rPA in  Nicotiana benthamiana  leaves  via  agro-infiltration. Several gene expression cassettes were designed, synthesized  in vitro , and then cloned into  Tobacco mosaic virus  RNA-based overexpression vector. Codon optimization, subcellular targeting, and the effect of attached Strep-tag II were assessed to identify conditions that maximized expression levels of the recombinant rPA in tobacco leaves. We found that codon-optimized rPA with N-terminal Strep-tag II that was aimed to the endoplasmic reticulum as target provided the highest amount of biologically active protein, i.e., up to ∼50 mg from per kilogram fresh weight leaf biomass in less than 1 week. Furthermore, the recombinant rPA was conveniently purified from inoculated leaf extracts by a one-step purification procedure  via  the Strep-tag II. The plant-made rPA was glycosylated with molecular mass of ∼45.0 kDa, and its  in vitro  fibrinolysis activity was equivalent to the commercial available rPA. These results indicate that the plant viral amplicon-based system offers a simple and highly effective approach for cost-effective large-scale production of recombinant rPA.

Production of Bioactive Recombinant Reteplase by Virus-Based Transient Expression System in Nicotiana benthamiana