Expanding the Genetic Code with Pyroglutamate

Abstract

Pyroglutamate (pGlu) is an amino acid found in a number of proteins including amyloid b-peptides associated with Alzheimer's disease and oconase, an anti-cancer agent. It forms via post-translational modifications of N-terminal glutamine (Gln) or glutamate (Glu) residues via cyclization. To facilitate research into the function of pGlu, we are expanding the genetic code of E. coli to include the modified amino acid via an amber suppressor system. To add pGlu to the code, we modified the archaeal RNA-dependent Gln biosynthetic pathway using a glutamyl-tRNA synthetase and the amidotransferase GatDE to synthesizepGlu on a mutant archaeal amber suppressor tRNA (tRNApGlu). The system is orthogonal in E. coli. The relevant genes were cloned into a pRSF-Duet vector and transformed into E. coli to determine if they can form pGlu-tRNApGlu in vivo. Read-through of an amber codon in a genetic message is being verified using an enhanced yellow fluorescence protein (eYFP) reporter system. Site-specific incorporation of pGlu into the protein will be validated by mass-spectrometry. Once established, we will use the system to test the role of the amino acid in RNaseA. This work was supported by a Research Corporation Cottrell College Science Award and Skidmore College.