Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an <em>Escherichia coli</em> 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.

Bead-Based Assay for Spatiotemporal Gene Expression Control in Cell-Free Transcription–translation Systems

“…If the canonical amino acid is still present within the system, it will be preferably incorporated instead of the noncanonical one [32]. Using the method proposed here, a first expression can be performed to incorporate the residual canonical amino acid into a dummy protein, followed by addition of the corresponding noncanonical amino acid in a second expression reaction.…”

From Superficial Damage to Invasion of the Nucleosome: Ranking of Morbidities by the Biosemiotic Depth Hypothesis

“…It is still uncertain when and where misincorporation can occur in vivo, though some research suggests that misincorporation of canavanine in place of arginine may happen in the mitochondrial ribosome causing normal protein synthesis to stall [257]. Because misincorporation of BMAA, for one, is blocked in many cases by editing taking place in the mitochoindria [258], [259], a minor level of excitotoxicity of non-coding BMAA, rather than its misincorporation into proteins [243], [254], is believed by some to be causing cell death and contributing to systemic autoimmunities [260], [261].…”

A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems

“…The residue specific incorporation of non-canonical amino acids into proteins is a far reaching application of cell-free transcription-translation systems. Non-canonical amino acids as a surrogate at canonically prescribed locations can lead to new or improved properties of proteins with potential applications in biochemistry, biotechnology and biomedicine (Worst et al, 2016(Worst et al, , 2015. However, for residue specific incorporation of non-canonical amino acids even small amounts of a corresponding canonical one will preferentially bind at the level of aminoacyl-tRNA-synthetases (Worst et al, 2016).…”

“…Non-canonical amino acids as a surrogate at canonically prescribed locations can lead to new or improved properties of proteins with potential applications in biochemistry, biotechnology and biomedicine (Worst et al, 2016(Worst et al, , 2015. However, for residue specific incorporation of non-canonical amino acids even small amounts of a corresponding canonical one will preferentially bind at the level of aminoacyl-tRNA-synthetases (Worst et al, 2016). Experiments by Hopfield have shown that a mechanism termed kinetic proofreading leads to a discrimination of up to 1/1000 against the incorrect amino acid (Hopfield, 1974;Hopfield et al, 1976).…”

“…Escherichia coli based expression systems are prepared "devoid" of amino acids, however, still there is a high level of remaining L-lysine, compared to other amino acids (Worst et al, 2016). In the following we will discuss methods for the removal of canonical amino acids, in particular L-lysine.…”