High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites
ABSTRACT 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.
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- "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 "
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. DOI:10.1016/j.jbiotec.2014.02.009 · 2.88 Impact Factor
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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 · 8.04 Impact Factor
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ABSTRACT: Escherichia coli-based cell extract is a vital component of inexpensive and high-yielding cell-free protein synthesis reactions. However, effective preparation of E. coli cell extract is limited to high-pressure (French press-style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. Here we report the viability of E. coli cell extract prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples. Specifically, we assessed the low-capital-cost lysis techniques of: (i) sonication, (ii) bead vortex mixing, (iii) freeze-thaw cycling, and (iv) lysozyme incubation to prepare E. coli cell extract for cell-free protein synthesis (CFPS). We also used simple shake flask fermentations with a commercially available E. coli strain. In addition, RNA polymerase was overexpressed in the E. coli cells prior to lysis, thus eliminating the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high-yielding E. coli-based extract was prepared using equipment requiring a reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing.BioTechniques 09/2012; 53(3):163-74. DOI:10.2144/0000113924 · 2.75 Impact Factor