Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain.
We recently developed a practical protocol for preparing proteins bearing stereo-selectively (13)C-methyl labeled leucines and valines, instead of the commonly used (13)C-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.
Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.
Yohei
Y
Miyanoiri
Center for Advanced Biotechnology and Medicine, Rutgers University-Robert Wood Johnson Medical School, 679 Hoes Lane West, Piscataway, NJ, 08854, USA.
Yojiro
Y
Ishida
Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.
Mitsuhiro
M
Takeda
Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, 192-0397, Japan.|SAIL Technologies, Inc., 2008-2 Wada, Tama, Tokyo, 206-0001, Japan.
Tsutomu
T
Terauchi
Center for Advanced Biotechnology and Medicine, Rutgers University-Robert Wood Johnson Medical School, 679 Hoes Lane West, Piscataway, NJ, 08854, USA.
Masayori
M
Inouye
Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan. kainosho@tmu.ac.jp.|Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, 192-0397, Japan. kainosho@tmu.ac.jp.
Masatsune
M
Kainosho
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D007532:Isoleucine / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling; D007930:Leucine / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D014633:Valine / Q000737:chemistry* / Q000378:metabolism
D011506:Proteins; D007532:Isoleucine; D007930:Leucine; D014633:Valine
Auxotrophic E. coli; Large protein; Methyl–methyl NOEs; stereo-specifically methyl labeled Ile/Leu/Val