Abstract
We recently developed a practical protocol for preparing proteins bearing stereo-selectively 13C-methyl labeled leucines and valines, instead of the commonly used 13C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.
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Acknowledgments
This research was supported by the Platform Project for Supporting in Drug Discovery and Life Science Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and Japan Agency for Medical Research and development (AMED). M. K. was supported by JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (21121002 and 26119005). Y. M. was supported by JSPS KAKENHI Grants-in-Aid for Young Scientists (B) (23770111 and 25840021) and a Grant-in-Aid for Scientific Research (C) (15K06966). M. T. was supported by a JSPS KAKENHI Grant-in-Aid for Young Scientists (B) (23770109), a Grant-in-Aid for Scientific Research (C) (25440018), and a grant from the Kurata Memorial Hitachi Science and Technology Foundation. We would like to thank Dr. Tugarinov, of the University of Maryland, for providing the MSG gene and his advice regarding MSG protein production.
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TT. was an employee of SAIL Technologies, Inc. during the present research, and M.K. is a Research Advisor for the company.
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Yohei Miyanoiri and Yojiro Ishida have contributed equally to this work.
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Miyanoiri, Y., Ishida, Y., Takeda, M. et al. Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain. J Biomol NMR 65, 109–119 (2016). https://doi.org/10.1007/s10858-016-0042-0
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DOI: https://doi.org/10.1007/s10858-016-0042-0