Modification of Aminoacyl tRNA Synthetase in Order to Incorporate An Unnatural Amino Acid

Abstract

Proteins allow daily processes in the cell to occur. A protein consists of amino acids. There are twenty natural amino acids coded for in the DNA of organisms. The natural amino acids can be modified to form unnatural amino acids (UAAs). UAAs have useful characteristics when inserted into a protein of a cell, like the ability of fluorescence and the ability to undergo unique reactions. For an UAA to be incorporated into a protein, it must be bound to a transport RNA molecule by an enzyme called aminoacyl tRNA synthetase (aaRS). An existing aaRS was modified in E. Coli bacterial cells to incorporate 3-(2-pyridyl)-L-Alanine since it has metal-binding capabilities. Once incorporated, the UAA could act as a sensor for a metal, making it useful to environmental fields. The aaRS was randomly mutated using saturation mutagenesis at sites L32, V65, W108, G158, A159. The cells were run through a positive screen to determine if the mutated aaRS incorporated the UAA into a green fluorescent protein, which glowed if the UAA was inserted. The results of the positive screen showed several mutated aaRSs (2,4,7,8) incorporated 3-(2-pyridyl)-L-Alanine, while other mutants (2,5,6,7,8,9) also/instead incorporated the p-cyanophenylalanine amino acid that the original aaRS was designed to select