Improving the Synthesis of an Unnatural Fluorescent Amino Acid

The long-term goal of this project is to more efficiently synthesize an unnatural fluorescent amino acid, 3-[7-nitro-2,1,3-benzoxadiazol-4-yl]-L-alanine, diazole for short. This fluorescent amino acid can be incorporated into a transparent protein, thus creating a glow-in-the-dark protein. A biocatalysis pathway is being explored to synthesize this diazole amino acid. Specifically, we used the enzyme glutathione S-transferase from the cyanobacterium T. elongatus to catalyze the key nucleophilic aromatic substitution reaction. The gene for a His-tagged variant of this enzyme was inserted into a plasmid which was used to transform E. coli. Following protein expression, the cells were lysed and the enzyme was isolated using a nickel chelate column. Enzyme kinetics are monitored using LC-MS