Miro Pastrnak

A New Orthogonal Suppressor tRNA/Aminoacyl-tRNA Synthetase Pair for Evolving an Organism with an Expanded Genetic Code

Several steps have been completed toward the development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are orthogonal to all Escherichia coli endogenous tRNA/synthetase pairs, followed by directed evolution of the orthogonal aminoacyl-tRNA synthetases to alter their amino-acid specificities. A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair in E. coli has been derived from the Saccharomyces cerevisiae tRNAAsp and aspartyl-tRNA synthetase, and the in vitro and in vivo characteristics of this pair were determined. Two different antibiotic resistance selections were compared using this novel pair in an effort to develop a tunable positive selection for a mutant synthetase capable of charging its cognate suppressor tRNA with an unnatural amino acid.

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.