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Site-Specific Incorporation of Selenocysteine Using an Expanded Genetic Code and Palladium-Mediated Chemical Deprotection

Liu, Jun, Zheng, Feng, Cheng, Rujin, Li, Shanshan, Rozovsky, Sharon, Wang, Qian, Wang, Lei
Journal of the American Chemical Society 2018 v.140 no.28 pp. 8807-8816
Escherichia coli, catalysts, enzyme activity, genetic code, glutathione peroxidase, human health, humans, palladium, selenocysteine, selenoproteins, stop codon, thioredoxins, translation (genetics)
Selenoproteins containing the 21ˢᵗ amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The distinct low pKₐ, high reactivity, and redox property of Sec also afford unique routes to protein modification and engineering. However, natural Sec incorporation requires idiosyncratic translational machineries that are dedicated to Sec and species-dependent, which makes it challenging to recombinantly prepare selenoproteins with high Sec specificity. As a consequence, the function of half of human selenoproteins remains unclear, and Sec-based protein manipulation has been greatly hampered. Here we report a new general method enabling the site-specific incorporation of Sec into proteins in E. coli. An orthogonal tRNAᴾʸˡ-ASecRS was evolved to specifically incorporate Se-allyl selenocysteine (ASec) in response to the amber codon, and the incorporated ASec was converted to Sec in high efficiency through palladium-mediated cleavage under mild conditions compatible with proteins and cells. This approach completely obviates the natural Sec-dedicated factors, thus allowing various selenoproteins, regardless of Sec position and species source, to be prepared with high Sec specificity and enzyme activity, as shown by the preparation of human thioredoxin and glutathione peroxidase 1. Sec-selective labeling in the presence of Cys was also demonstrated on the surface of live E. coli cells. The tRNAᴾʸˡ-ASecRS pair was further used in mammalian cells to incorporate ASec, which was converted into Sec by palladium catalyst in cellulo. This robust and versatile method should greatly facilitate the study of diverse natural selenoproteins and the engineering of proteins in general via site-specific introduction of Sec.