Protein Engineering by In Vivo Incorporation of Non-Natural Amino Acids: Control of Incorporation of Methionine Analogues by Methionyl-tRNA Synthetase

The incorporation of non-natural amino acids is an important strategy for engineering novel chemical and physical properties into natural and artificial proteins. The incorporation of amino acids into proteins in vivo is controlled in large part by the aminoacyl-tRNA synthetases (AARS). We have measured kinetic constants for in vitro activation of a set of methionine analogues by methionyl-tRNA synthetase (MetRS) via the ATP–PPi exchange reaction. Activation of methionine analogues in vitro correlates well with the ability of these analogues to support protein synthesis in vivo, substantiating the critical role of the AARS in controlling the incorporation of non-natural amino acids into proteins. Methionine analogues with kcat/Km values 2000-fold lower than those for methionine can support synthesis of a typical target protein (mDHFR) under standard conditions of protein expression. The kinetic constants correlate well with observed protein yields from a conventional bacterial expression host, indicating that the MetRS activity of the host can control the level of protein synthesis under certain conditions. Furthermore, increasing the MetRS activity of the bacterial host results in increased protein synthesis in media supplemented with the methionine analogues homoallylglycine and norleucine. These results suggest new strategies for incorporation of non-natural amino acids via manipulation of the AARS activity of a bacterial host.