Expanding the scope of templated macromolecular synthesis in vivo: The incorporation of methionine analogues into proteins in vivo by altering the methionyl-tRNA synthetase activity of a bacterial expression host

• Main Author: Kiick, Kristi Lynn
• Language: ENG
• Published: ScholarWorks@UMass Amherst 2001
• Subjects: Polymers|Biochemistry
• Online Access: https://scholarworks.umass.edu/dissertations/AAI3012149

The incorporation of non-natural amino acids is an important strategy for engineering novel chemical and physical properties into natural and artificial proteins. The in vivo incorporation of non-natural amino acids is controlled by the aminoacyl-tRNA synthetases (aaRS), the class of enzymes that catalyzes the covalent attachment of amino acids to their cognate tRNAs. The correlation between the incorporation of methionine analogues into proteins in vivo, and activation of the analogues by methionyl-tRNA synthetase (MetRS) in vitro has therefore been investigated. Kinetics constants for the in vitro activation of a set of methionine analogues, 2–13, have been measured 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 (DHFR) under standard conditions of protein expression employing a conventional bacterial host. The kinetics constants also correlate well with observed protein yields from a conventional expression host, indicating that the MetRS activity of the host can control the level of protein synthesis under certain conditions. Overexpression of MetRS in a bacterial host was investigated as a method to increase the number of methionine analogues that can be incorporated into proteins in vivo. Conventional bacterial hosts are able to utilize analogues 2 (homoallylglycine), 3 (homopropargylglycine), and 9 (norleucine) during protein synthesis. Equipping a bacterial host with extra copies of the gene encoding wild-type MetRS permits incorporation, under a set of standard conditions, of additional unsaturated methionine analogues 5 (trans-crotylglycine) and 11 (2-butynylglycine). Increasing the concentration of analogue in the culture medium from the “normal” value of 20 mg/L to 500 mg/L results in the incorporation of four additional analogues, 4 (cis-crotylglycine), 7 (2-aminoheptanoic acid), 8 (norvaline), and 12 (allylglycine). Assessment of the level of replacement of methionine by the analogues via amino acid analysis and N-terminal sequencing indicates that the extent of replacement is roughly correlated with the kcat/Km values for activation of the analogue. (Abstract shortened by UMI.)