A Study of Protein Expression and Enzyme Selectivity Using Unnatural Amino Acids

Abstract

A study of substrate selectivity of aminoacyl-tRNA synthetases, from both bacterial and eukaryotic organisms, using unnatural amino acids is described and discussed in this thesis.

The first part of this thesis involved an investigation of the substrate selectivity of bacterial (E. coli) aminoacyl-tRNA synthetases, by protein synthesis using unnatural amino acids via bacterial (E. coli) wild-type machinery. The levels of unnatural amino acid incorporation obtained are discussed in relation to the substrate selectivity of aminoacyl-tRNA synthetases. This work clearly shows that the E. coli TrpRS (Chapter One), the E. coli PheRS (Chapter Two) and the E. coli TyrRS (Chapter Three) display some degree of substrate promiscuity.

An HPLC technique has been used to determine the relative rate of unnatural amino acid activation by E. coli TyrRS for a series of meta- and ortho-substituted tyrosines. This work, which is discussed in Chapter Four, shows that the reaction rate of aminoacylation is significantly affected by the substituent on the aromatic ring of tyrosine.

The other part involved a study of the substrate selectivity of eukaryotic aminoacyl-tRNA synthetase. Although the original work, which was conducted to investigate the substrate selectivity of eukaryotic PheRS and TyrRS by incorporating the unnatural amino acids into protein with a eukaryotic protein synthesis system and is discussed in Chapter Five, had been unsuccessful, an investigation of the human TyrRS with a series of meta- and ortho-substituted tyrosines using the ITC technique, which is discussed in Chapter Six, shows some degree of substrate promiscuity of human TyrRS. In addition, a selectivity comparison made between the bacterial (E. coli) and the eukaryotic (human) TyrRSs, which is also discussed in Chapter Six, shows that there is difference between these two enzymes regarding substrate selectivity, resulting in the discovery of two organic compounds that could potentially be developed as antimicrobial agents. Show more