title: Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.
abstract: The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.
authors: Department of Experimental Physics, Saarland University.
authors: Emanuel G
authors: EG
authors: Worst
authors: Institute of Chemistry, Technische Universität Berlin.
authors: Matthias P
authors: MP
authors: Exner
authors: Institute of Chemistry, Technische Universität Berlin.
authors: Alessandro
authors: A
authors: De Simone
authors: Department of Experimental Physics, Saarland University.
authors: Marc
authors: M
authors: Schenkelberger
authors: School of Physics and Astronomy, University of Minnesota.
authors: Vincent
authors: V
authors: Noireaux
authors: Institute of Chemistry, Technische Universität Berlin.
authors: Nediljko
authors: N
authors: Budisa
authors: Department of Experimental Physics, Saarland University; albrecht.ott@physik.uni-saarland.de.
authors: Albrecht
authors: A
authors: Ott
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D000604:Amino Acyl-tRNA Synthetases