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Expanding the genetic code with pyroglutamate
Author(s) -
Walsh Matthew,
Benhaim Mark,
Sheppard Kelly
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.614.2
Subject(s) - transfer rna , glutamine , genetic code , biochemistry , mutant , glutamine amidotransferase , stop codon , amino acid , chemistry , biology , rna , microbiology and biotechnology , gene
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 (tRNA pGlu ). The ability of the archaeal GluRS and mutant GatDE to use tRNA pGlu as a substrate in vitro was investigated using gel‐shift assays and kinetic studies. The genes for tRNA pGlu and the two aforementioned enzymes were cloned into a pRSF‐Duet vector and transformed into E. coli to determine if they can form pGlu‐tRNA pGlu 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.