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

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 con...