A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
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A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo even with amino acid types that are central to the metabolic pathways, such as glutamine. The sequential backbone resonance assignment of the Neh2 domain of Nrf2 transcriptional factor, an intrinsically disordered protein with high spectral degeneracy, was achieved using this labeling method.
KeywordsAmino acid selective isotope labeling Intrinsically disordered protein Neh2 15N-glutamate labeling 15N-glutamine labeling
We would like to thank Dr. Stefan Bagby of the University of Bath for valuable discussion and critical reading of the manuscript. We would also like to thank Dr. Yasutake Katoh for the use of the plasmid, Drs. Yu Wang and Priscilla T. Y. Leung of the Genome Research Centre in the University of Hong Kong for mass spectrometry analysis, and the Colorado State University Macromolecular Resources Facility for the help in the initial mass spectrometry analysis. This work was supported in part by grants-in-aid from JST-ERATO and the Ministry of Education, Culture, Sports, Science, and Technology. T.T. was supported by the 21st Century COE Program and the Protein 3000 project.
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