EYFP reporter system for pyroglutamate incorporation

Abstract

Pyroglutamate (pGlu) is an amino acid formed from modification of glutamine in proteins like β‐amyloid peptides associated with Alzheimer's disease and the anti‐cancer agent, onconase. To allow for in‐depth studies into the role pGlu plays in proteins and its relationship to diseases like Alzheimer's, it would be beneficial to synthesize polypeptides with pGlu incorporated during translation (i.e., expand the genetic code to include pGlu). We have developed a means to synthesize pGlu on transfer RNA by modifying the archaeal system for tRNA‐dependent glutamine formation and placed the system into E. coli to decode amber stop codons (UAG) with pGlu. To determine if there is site‐specific 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. Position 14 in the protein is normally proline, a chemical analog of pGlu. We have mutated efyp to have an amber, opal, or ochre stop codon at codon 14. After optimizing eYFP overproduction, we will determine if our modified archaeal system in E. coli allows for specific decoding of the amber stop codon in eyfp with pGlu.