Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Here we report the development of a baculovirus-based delivery system that enables the efficient incorporation of unnatural Amino acids into proteins in mammalian cells. We have exploited the large cargo-capacity (>30 kb) and stability of the double-stranded DNA genome of baculovirus to deliver to a variety of cell types all of the components required to genetically incorporate novel Amino acids. These include the engineered tRNA/Aminoacyl-tRNA Synthetase pair and the nonsense mutant of the target gene. Mammalian cell transduction efficiency of baculovirus was significantly improved by incorporating genetic elements from mammalian viruses. Two polyspecific tRNA/Aminoacyl-tRNA Synthetase pairs were inserted into this expression system, enabling the site-specific incorporation of a variety of unnatural Amino acids with novel chemical and biological properties into proteins.