Liutkus, Mantas2016-06-272016-06-27b39905408http://hdl.handle.net/1885/104840A new method for the production of peptides through biological expression was developed, utilising lactonisation-prone chlorinated amino acids for latent peptide bond digestion. Incorporation of halogenated amino acids into proteins is possible due to the inherent inability of the biological synthetic machinery to discriminate against compounds structurally similar to the natural substrates. As isoleucine, leucine and valine are primarily recognised by size exclusion, all isosteres with a methyl group replaced by similarly-sized chlorine are mistaken as substrates, and the ability of halogens to mimic the bonding interactions of sulfur enabled the design of a chlorinated and a brominated analogue of methionine. Amino acids with chlorine at the 4-position, incorporated into proteins in place of isoleucine, leucine or methionine during cell-free protein expression, were found to trigger cleavage of the proximal peptide bond on the C-terminal side at elevated temperatures. The reaction mechanism is similar to that of cyanogen bromide induced cleavage, driven by the formation of a highly favourable 5-membered ring. This presents the first case of heat induced proteolysis, resulting in almost instantaneous peptide bond cleavage through exposure to 100 degrees Celsius in water, and avoids the need for toxic, expensive or sensitive external agents. When encoded in a fusion protein, the chlorinated residues can be used for rapid separation of the fusion partners. The utility of the method was demonstrated through the preparation of small peptides human gastrin releasing peptide prohormone, cholecystokinin prohormone and oxytocin, lacking isoleucine, leucine and methionine, respectively, expressed as fusion proteins. Through simultaneous replacement of two amino acids, deuterated and fluorinated analogues of the peptides were also prepared. The method was then expanded to prepare more complicated targets. Homologous substitution of leucine with isoleucine, and vice versa, enabled the preparation of a small protein aprotinin. A crystal structure of the mutated aprotinin demonstrated that the protein structure was not affected by the substitution, thus establishing that interchange of similar amino acids can be used to overcome sequence limitations. In stark contrast, amino acids with chlorine at the 3-position are resistant to high temperature. Through the ability to substitute valine or isoleucine during protein expression, the chlorides were incorporated into aprotinin, thus establishing a method for the preparation of proteins of essentially unlimited size containing 3-chloro amino acids. Crystal structures of the chlorinated aprotinin showed that the amino acid analogues are not inherently detrimental to protein structure and can lead to stable proteins, while the preparation through the expression of heat-labile fusion proteins juxtaposed the differences in reactivity between amino acids halogenated at the 3- and 4-positions.enchlorinated amino acidspeptide productionlactamisationlactamizationcell-free protein expression201610.25911/5d778b6b50f49