Abstract
This chapter describes protocols for preparing 15N-labeled proteins (ubiquitin is used as an example) using Escherichia coli cells (with purification) and the wheat germ cell-free system (without purification). A comparison of 1H-15N heteronuclear single-quantum coherence (HSQC) spectra of yeast ubiquitin prepared using each method indicates that this wheat germ cell-free system may be used for rapid nuclear magnetic resonance analyses of proteins without purification.
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Acknowledgments
The author thanks Drs. Y. Endo and T. Sawasaki for providing the pEU3b plasmid and useful discussions. I also thank Mr. T. Ogasawara and Dr. H. Morita for useful discussions, Dr. T. Sakamoto for preparation of 15N-labeled ubiquitin from E. colicells, Mss. M. Sugai and C. Komatsu for preparing plasmids pET-24a/ubiquitin and pEU3b/ubiquitin, and Ms. R. Tanaka for the preparation of 15N-labeled ubiquitin with the wheat germ cell-free system. This work was supported in part by a grant from National Project on Protein Structural and Functional Analyses.
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Kohno, T. (2005). Production of Proteins for NMR Studies Using the Wheat Germ Cell-Free System. In: Zanders, E.D. (eds) Chemical Genomics. Methods in Molecular Biology™, vol 310. Humana Press. https://doi.org/10.1007/978-1-59259-948-6_12
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DOI: https://doi.org/10.1007/978-1-59259-948-6_12
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