Abstract
Pyroglutamate (pGlu) forms via the cyclization of N‐terminal glutamine (Gln) or glutamate (Glu) residues in a wide‐range of proteins including amyloid β‐peptides associated with Alzheimer's disease and oconase, an anti‐cancer agent member of the RNaseA superfamily. To facilitate research into the role pGlu plays in these proteins, we are expanding the genetic code of E. coli to include the modified amino acid by reassigning the amber stop codon to pGlu. To include pGlu, we are modifying the archaeal RNA‐dependent Gln biosynthetic pathway [glutamyl‐tRNA synthetase (GluRS) and the amidotransferase GatDE] to synthesize pGlu on a mutant archaeal amber suppressor tRNA (tRNApGlu). The ability of the archaeal GluRS and mutant GatDE to use tRNApGlu as a substrate in vitro was investigated using gel‐shift assays and kinetic studies. The genes for tRNApGlu and the two aforementioned enzymes were cloned into a pRSF‐Duet vector and transformed into E. coli to determine if they can form pGlu‐tRNApGluin 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.
Published Version
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