Production and downstream processing optimization of plant-made Osmotin and its functional analysis

Abstract Plant-made therapeutic proteins are getting acceptance because of the cost-effective production that displays equal efficacy to the more established platform such as mammalian or bacterial-based production systems. This study demonstrates that the stable expression of recombinant osmotin (rOSM) expressed and purified in large amounts is generally regarded as safe (GRAS) lettuce leaf tissues. In this study, we designed experiments to explore a plant-based system for the expression of rOSM known for exhibiting antifungal activity and stress tolerance. The codon-optimized osmotin sequences for higher expression in lettuce were added with 6xHistag and ER retention signal peptide and three independent transgenic lines were generated and screened for transgene expression with PCR. The protein extraction was optimized considering the impact of ultrasonication, PIC, and tween-20 impact on total protein extraction. Immunoblot analysis confirmed the induction of ~ 28.5 kDa recombinant fusion protein and ELISA quantitation was carried out to confirm the expression level of 127 mg/kg fresh weight (FW) in lettuce leaf tissues. The method has been developed to purify recombinant proteins from leaf tissues with relatively convenient manual techniques for histidine-tagged protein with final product purity of > 90%. The functional analysis of purified proteins exhibited antifungal activity was verified using two human pathogenic fungal strains. In addition, the expression of rOSM made this plant more versatile to tolerate adverse environmental conditions when compared with wild-type cultivars. In this study, we demonstrated that the lettuce plant can produce a high level of functional protein and therefore is a promising production system for therapeutic purposes. Additionally, we outline the dual functionality of expressing osmotin for the synthesis of therapeutic proteins, thereby endowing engineered plants with enhanced stress tolerance.