Abstract
Genetic code expansion allows unnatural (non-canonical) amino acid incorporation into proteins of interest by repurposing the cellular translation machinery. The development of this technique has enabled site-specific incorporation of many structurally and chemically diverse amino acids, facilitating a plethora of applications, including protein imaging, engineering, mechanistic and structural investigations, and functional regulation. Particularly, genetic code expansion provides great tools to study mammalian proteins, of which dysregulations often have important implications in health. In recent years, a series of methods has been developed to modulate protein function through genetically incorporated unnatural amino acids. In this review, we will first discuss the basic concept of genetic code expansion and give an up-to-date list of amino acids that can be incorporated into proteins in mammalian cells. We then focus on the use of unnatural amino acids to activate, inhibit, or reversibly modulate protein function by translational, optical or chemical control. The features of each approach will also be highlighted.
Highlights
Knowledge of protein function is of pivotal importance to life science research
Genetic code expansion has matured into a technique that can be routinely used in mammalian systems
Controlling protein function is currently mostly achieved by translation, light, and small molecules
Summary
Knowledge of protein function is of pivotal importance to life science research. It can guide conventional drug development programmes and lead to novel strategies to address currently non-targetable systems [1,2,3]. Targeting the protein by small-molecule inhibition is often not possible in these cases, as protein homologues will be affected To tackle this problem, over the last two decades there has been a drive to develop and refine the technique of genetic code expansion which allows researchers to exploit the cellular translation machinery for site-specific incorporation of unnatural (non-canonical) amino acids into target proteins [4,5,6,7,8,9,10,11,12,13,14]. The tRNAPyl naturally recognises the UAG codon and engineering of this tRNA is not needed This pair is orthogonal in both E. coli and mammalian cells; it facilitates the engineering of PylRS in E. coli and subsequently using the engineered PylRS mutant for incorporation of the designated unnatural amino acid in mammalian systems. Amber suppression allows a more stringent control, as the background activity (if any) can be further
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