Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.
"While hundreds of different non-canonical amino acids have been translationally incorporated into proteins, much of this work has primarily been carried out in vivo (reviewed in (Liu and Schultz, 2010)). Recent advancements in CFPS systems have made in vitro production of proteins containing unnatural amino acids via site-specific incorporation an attractive alternative to in vivo methods (Albayrak and Swartz, 2013a, 2013b; Arthur et al., 2013; Bundy and Swartz, 2010; Goerke and Swartz, 2009; Hong et al., 2014; Loscha et al., 2012; Ozawa et al., 2012). These studies have focused on the site-specific incorporation of unnatural amino acids via the use of engineered translation system components, and the orthogonal suppression of stop-codons (Liu and Schultz, 2010) or "
[Show abstract][Hide abstract] ABSTRACT: Residue specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid-depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.
Journal of Biotechnology 05/2014; 178(1). DOI:10.1016/j.jbiotec.2014.02.009 · 2.87 Impact Factor
"Furthermore, this approach allowed efficient 15N-labeling of individual proteins in selective (58,59) and combinatorial (38,39) labeling schemes, providing a route to NMR resonance assignments of samples of limited solubility and stability. Finally, the cell-free approach is uniquely suited for the incorporation of unnatural amino acids (31), in the present work affording the facile incorporation of the unnatural amino acid Bpa for photo-crosslinking. This method may present a useful tool to probe structures of larger replisomal complexes in the future. "
[Show abstract][Hide abstract] ABSTRACT: A complex of the three (αεθ) core subunits and the β2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β2 replicase complex with primer-template DNA combine all available structural data.
Nucleic Acids Research 04/2013; 41(10). DOI:10.1093/nar/gkt162 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Just as synthetic organic chemistry once revolutionized the ability of chemists to build molecules (including those that did not exist in nature) following a basic set of design rules, cell-free synthetic biology is beginning to provide an improved toolbox and faster process for not only harnessing but also expanding the chemistry of life. At the interface between chemistry and biology, research in cell-free synthetic systems is proceeding in two different directions: using synthetic biology for synthetic chemistry and using synthetic chemistry to reprogram or mimic biology. In the coming years, the impact of advances inspired by these approaches will make possible the synthesis of nonbiological polymers having new backbone compositions, new chemical properties, new structures, and new functions.
Current Opinion in Biotechnology 04/2012; 23(5):672-8. DOI:10.1016/j.copbio.2012.02.002 · 7.12 Impact Factor
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