Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein using Escherichia coli cell extract-based CF. This method takes advantage of endogenous metabolic conversions to generate SI-labeled asparagine, glutamine, cysteine, and tryptophan, which are much more expensive than the other 16 kinds of SI-labeled amino acids, from inexpensive sources, such as SI-labeled algal amino acid mixture, SI-labeled indole, and sodium sulfide, during the CF reaction. As compared with the conventional method employing 20 kinds of SI-labeled amino acids, highly enriched uniform SI-labeling with similar labeling efficiency was achieved at a greatly reduced cost with the newly developed method. Therefore, our method solves the cost problem of the SI labeling of proteins using the CF.
"To deal with this problem, we used the E. coli S30 cell-free translation system lacking a specific amino acid. We considered that, in some cases, we should eliminate the activities of the enzymes involved in the biosynthesis of the amino acids removed from the cell-free extract (41). Indeed, a minor band appeared under the conditions without cysteine (Supplementary Figure S4B, lane 2). "
[Show abstract][Hide abstract] ABSTRACT: At earlier stages in the evolution of the universal genetic code, fewer than 20 amino acids were considered to be used. Although this notion is supported by a wide range of data, the actual existence and function of the genetic codes with a limited set of canonical amino acids have not been addressed experimentally, in contrast to the successful development of the expanded codes. Here, we constructed artificial genetic codes involving a reduced alphabet. In one of the codes, a tRNA(Ala) variant with the Trp anticodon reassigns alanine to an unassigned UGG codon in the Escherichia coli S30 cell-free translation system lacking tryptophan. We confirmed that the efficiency and accuracy of protein synthesis by this Trp-lacking code were comparable to those by the universal genetic code, by an amino acid composition analysis, green fluorescent protein fluorescence measurements and the crystal structure determination. We also showed that another code, in which UGU/UGC codons are assigned to Ser, synthesizes an active enzyme. This method will provide not only new insights into primordial genetic codes, but also an essential protein engineering tool for the assessment of the early stages of protein evolution and for the improvement of pharmaceuticals.
Nucleic Acids Research 08/2012; 40(20). DOI:10.1093/nar/gks786 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Localized vibrational mode (LVM) studies in neutron irradiated oxygen-rich silicon have been carried out in order to investigate the origin of certain bands appearing in the spectra after heat treatment. The investigation was mainly focused on two satellite lines at 840 cm <sup>-1</sup> and 825 cm<sup>-1</sup> observed on either side of the 829 cm<sup>-1</sup> band of VO above 250°C and 350°C respectively, upon 15 min isochronal annealings. Theoretical analysis supports the identification of the two satellites with V<sub>2</sub>O and V<sub>2 </sub>O<sub>2</sub> defects respectively
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