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An Efficient Protocol For Incorporation Of An Unnatural Amino Acid In Perdeuterated Recombinant Proteins Using Glucose-Based Media, Vincenzo Venditti, Nicolas L. Fawzi, G. Marius Clore

Vincenzo Venditti

The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR field. Here, we describe a novel protocol for incorporating unnatural amino acids into fully deuterated proteins using glucose-based media (which are relevant to the production, for example, of amino acid-specific methyl-labeled proteins used in the study …