Alshengeti, SondosSondosAlshengeti2022-05-162022-05-162020 the A2020http://hdl.handle.net/10197/12881The use of non-natural amino acids in protein engineering has become a valuable tool for studying protein functions, for selective chemical modification of proteins, and for improving protein function for biotechnology and medical applications. In this Master Thesis, a Green Fluorescent Protein Binding nanobody (GBP1) has been modified by introducing an azido group containing amino acid, p-azidophenylalanine, into its binding site (Arg 35) with the aim of constructing an activatable GBP1 nanobody variant for time-resolved antigen binding studies. Subsequent steric blocking of the GBP1 substrate recognition site will be achieved by conjugating a PEG moiety linked to a photocleavable group that is conjugated to the azide of pAzF. Site-specific incorporation was performed using the amber suppression strategy, with a polyspecific engineered TyrRS from M. jannaschii and its cognate suppressor tRNATyr. Protein yields were 0.5 mg/L of bacterial cell culture and the identity of the purified protein was assessed by mass spectrometry. In the second part of this work, attempts have been made to produce and purify the catalytic domain of an engineered pyrrolysyl-tRNA-synthetase from M. barkeri capable of recognizing histidine analogues with the intention of solving its X-ray structure. The availability of a crystal structure would facilitate the rational modification of the active site of the enzyme, for the generation of a novel pyrrolysyl-tRNA-synthetase capable of recognizing N- heterocyclic-carbene-alanine derivatives for incorporation into metalloenzyme oxidoreductases in place of catalytic histidine residues. Unfortunately, the produced protein was prone to aggregation under all test conditions and no soluble protein could be isolated.enProtein engineeringNon-natural amino acidsAmber suppressionPyrrolysyl-tRNA-synthetaseMaster Thesis2021-12-09https://creativecommons.org/licenses/by-nc-nd/3.0/ie/