Premium
eYFP Reporter System for Pyroglutamate Incorporation
Author(s) -
Calzini Miles,
Zhen Stephanie,
Sheppard Kelly
Publication year - 2015
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.29.1_supplement.892.13
Subject(s) - glutamine , transfer rna , chemistry , stop codon , amino acid , translation (biology) , release factor , proline , biochemistry , rna , gene , messenger rna
Pyroglutamate (pGlu), a chemical analog of proline, forms via cyclization of amino‐terminal glutamine residues in proteins like b‐amyloid peptides associated with Alzheimer's disease and the anti‐cancer agent, onconase. To study the role pGlu plays in proteins and its relationship to diseases like Alzheimer's, it would be useful to synthesize proteins with pGlu incorporated during translation . We have developed a means to synthesize pGlu on an amber suppressor transfer RNA by modifying the archaeal route for tRNA‐dependent glutamine formation and placed the system into E. coli to decode amber stop codons (UAG) with pGlu. To verify incorporation of pGlu at amber stop codons, we have selected the enhanced yellow fluorescent protein (eYFP) as our reporter protein and codon 14 for pGlu incorporation as that position is normally proline in eYFP. We have mutated codon 14 in efyp to an amber stop codon. After optimizing eYFP overproduction in the presence of the plasmid encoding the pGlu pathway, we are testing pGlu incorporation into eYFP. This work is supported by a Research Corporation Cottrell College Science Award and Skidmore College.