Translational Incorporation of Unsaturated Amino Acids

Abstract

In nature proteins are synthesised using twenty standard proteinogenic amino acids. The unique linear sequences of these monomeric building blocks dictate the structure and functionality of the translated protein. Misincorporation of unnatural amino acids into this biosynthetic process is rare due to the high fidelity of aminoacyl-tRNA synthetases. This thesis is concerned with the in vitro residue-specific incorporation of unnatural amino acids to produce proteins and peptides with increased functional diversity and potential applications in biotechnology.

The cellular lysate from E.coli is a common source of translational machinery for in vitro protein synthesis and has been shown to remove many commonly encountered protecting groups from α- amino acids. A chemically intractable unnatural amino acid has been supplied to this cellular extract and its facile deprotection then coupled to protein biosynthesis to produce highly modified protein. This method is not limited to only residue- specific incorporation but is also compatible with site-specific systems.

Post-translational oxidation of proteins has been implicated in the progression of various degenerative diseases. It is postulated that oxidation could also occur pre-translationally as a result of the misincorporation of oxidised amino acids during protein synthesis. To validate this concept the misincorporation of seven dehydro analogues for the aliphatic amino acids valine, leucine and isoleucine, have been evaluated during cell-free protein synthesis using native translational machinery. The dehydro analogues of valine and leucine as well as one of the isoleucine analogues were effective replacements with incorporation levels of at least 75%. Typically misincorporation rates for proteinogenic amino acids (i.e., leucine in place of isoleucine) are estimated to be 0.01%. Here the relative misincorporation observed for the dehydro amino acids was significantly greater than typical misincorporation, with rates as high at 0.6%.

The introduced alkene functionality of 4,5-dehydroisoleucine and 4,5-dehydroleucine amino acids has been utilised in a convenient method for site-specific protein backbone cleavage. Upon treatment with iodine these amino acids undergo iodo-lactonisation to give five- membered ring lactones that undergo rapid hydrolysis of the proximal carboxyl amide bond. This has been exploited for the rapid production of peptide hormones from a parent fusion protein expressed using cell-free protein synthesis. By incorporating unnatural amino acids into protein, functionality not represented by the twenty proteinogenic amino acids can be introduced and utilised for a variety of applications. A trifluoromethylalkene and three vinyl halides were found to be translational substitutes of leucine. The degree of substitution for two of the analogues was found to be greater than 75%. The elaboration of the modified protein by cross-coupling chemistry has been investigated.

Overall the work presented in this thesis sheds insight on the theory of pre-translational oxidation of proteins and their possible implications in degenerative diseases. The use of cell-free protein synthesis and subsequent site-specific cleavage of dehydro amino acids presents a rapid approach for the production of peptides and also has potential applications in broader areas of biotechnology. It describes the incorporation of functional vinyl halide amino acid derivatives at sites previously occupied by an aliphatic amino acid.