Abstract
Cell-free protein synthesis is suitable for stable-isotope labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium l-glutamate (l-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium d-glutamate (d-Glu system). The productivity of the d-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the 1H–15N HSQC spectrum of the uniformly stable-isotope labeled Ras protein, prepared with the d-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the d-Glu system. These results indicate that the d-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of proteins.
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Abbreviations
- CAT:
-
Chloramphenicol acetyltransferase
- HSQC:
-
Heteronuclear single quantum coherence
- Ras(Y32W)/d-Glu:
-
Ras(Y32W) protein produced by the d-Glu system
- Ras(Y32W)/KOAc:
-
Ras(Y32W) protein produced by the KOAc system
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Acknowledgements
We are grateful to Aya Kurosawa, Natsumi Suzuki, Yasuko Tomo, Yukiko Fujikura, Satoko Yasuda, Fumiko Hiroyasu, and Masaomi Ikari for their technical assistance. We thank Naohiro Kobayashi and Tadashi Tomizawa for help with the NMR analysis, and Jun Yokoyama for valuable suggestions and comments. We also thank Azusa Ishii, Kiyomi Yajima, and Tomoko Nakayama for expert secretarial assistance. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI) of the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Matsuda, T., Koshiba, S., Tochio, N. et al. Improving cell-free protein synthesis for stable-isotope labeling. J Biomol NMR 37, 225–229 (2007). https://doi.org/10.1007/s10858-006-9127-5
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DOI: https://doi.org/10.1007/s10858-006-9127-5