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

Abstract

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.genetic code | viral vector | nonstandard amino acid | orthogonal

Playing with the Molecules of Life

“…86 Additionally, orthogonal aaRS/tRNA technologies have been used to incorporate ncAAs into proteins in mammalian cell lines at gm/L scale employing transient expression methods. 100-102 Viral vectors have allowed the ncAA machinery to be delivered efficiently into primary cells, as well as tissues, 96, 103, 104 where it was used among other applications to monitor voltage-sensitive changes in response to membrane depolarization events in neural cells. 100 …”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…However, the application of this technology in mammalian cells is limited by the inherent drawbacks of transient transfection, including the relatively low transfection efficiency of certain cell types (e.g., neurons and embryonic stem cells) and the toxicity of transfection reagents. To overcome these challenges, an enhanced suppression system delivered by a hybrid baculovirus has been reported (Chatterjee et al 2013c). This baculovirus-based system significantly increases the incorporation efficiency of ncAA into proteins in mammalian cells and allows ncAAs to be encoded in embryonic stem cells, rat cardiac fibroblasts, and neurons.…”

Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNATyr Suppressor

“…18–20 Use of a baculovirus-based delivery system to deliver both the engineered tRNA and aaRS components to mammalian cells has also been successful. 21 Another strategy has been to manipulate interactions with other components of the protein translation system, including release factors in bacterial and mammalian cells, 18,22 and the elongation factor Tu in bacteria. 23,24 The orthogonality of M. jannaschii tRNA Tyr was improved by overexpression of prolyl-tRNA synthetase (ProRS), outcompeting an undesired interaction of engineered M. jannaschii TyrRS with Escherichia coli tRNA Pro .…”