Article

An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system

RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
Journal of Biomolecular NMR (Impact Factor: 3.14). 11/2010; 48(4):193-201. DOI: 10.1007/s10858-010-9455-3
Source: PubMed

ABSTRACT

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.

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    • "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). "
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