KSEF RDE: Next Generation Protein Expression Vectors Derived From Coffee Ringspot Virus

Therapeutic proteins (TPs), such as ZMapp and Rituxan, account for roughly half the market value of pharmaceuticals. This proposal addresses a significant constraint on current protein expression systems by facilitating protein expression in divergent cell types, which differ in their ability to generate post-translational protein variants, a key factor influencing the activity(ies) of TPs. Such variants are essential in development of "biobetters": peptides and proteins having enhanced characteristics over the original TP molecule. Screening and expression of novel biobetters would be accelerated were it possible to move a common protein expression system between diverse cell types (plant, insect, mammalian). Each eukaryotic expression system has different limitations and advantages of variable importance depending on the expressed protein and a single expression system for use in multiple cell types would allow the ready identification and exploitation of the optimal system. Our preliminary data suggests that a recently emerged threat to coffee production, Coffee ringspot virus (CoRSV), a nucleotropic pathogen, could provide a platform enabling construction of next-generation vector systems designed for use in a broad range of cell types. Existing vector systems are not optimized for polycistronic expression, or mobilization of expression cassettes between plant and animal cell cultures. Viruses such as CoRSV move between plant and animal cells in their natural cycle of replication and hence have evolved for optimal expression in a wide range of eukaryotic cells. Therefore, this proposal takes advantage of the natural proclivity for CoRSV protein expression in diverse eukaryotic cells by building an expression system that can be mobilized between different types of host cells. The proposed polycistronic expression system will be developed in collaboration with ParaTechs Corporation (Aim 1), and the genetic screens and Illumina sequencing with The Kentucky Tobacco Research & Development Center and the UK Plant and Soil Sciences Department (Aim 2).