Abstract

Co‐translational, site‐specific incorporation of unnatural amino acids (UAAs) by nonsense suppression has proven to be a powerful tool for investigating and engineering protein structure and functions. This methodology utilizes an engineered aminoacyl‐tRNA synthetase/tRNA (aaRS/tRNA) pair, orthogonal to the host cell, to deliver the UAA of interest in response to a nonsense codon. The ability to use this toolbox to investigate and manipulate protein function in human and other mammalian cells can be highly beneficial. The primary challenge is to efficiently co‐deliver all the genetic components, and a lack of understanding regarding optimal expression levels of these components. Viral vectors are ideal vehicles for delivery, potentially enabling application in a variety of mammalian cell lines, and even live tissues and animals.Using a unique viral delivery vector, and optimal expression of the genetic components, we have substantially enhanced UAA mutagenesis efficiency in mammalian cells. We identified the tRNA as the limiting component, while the traditional aaRS expression level was shown to be unfavorably high. The large and complex cargo could be efficiently packaged within our unique baculoviral delivery vector‐ showcasing the prowfess of this platform.

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