Journal of Biomolecular NMR

, Volume 13, Issue 4, pp 369–374 | Cite as

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

  • Natalie K. Goto
  • Kevin H. Gardner
  • Geoffrey A. Mueller
  • Randall C. Willis
  • Lewis E. Kay
Article

Abstract

A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce (1H-δ methyl)-leucine and (1H-γ methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≈100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] α-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-δ1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.

deuteration α-ketobutyrate α-ketoisovalerate methyl protonation 

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Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Natalie K. Goto
    • 1
  • Kevin H. Gardner
    • 3
  • Geoffrey A. Mueller
    • 2
  • Randall C. Willis
    • 2
  • Lewis E. Kay
    • 3
  1. 1.Department of BiochemistryUniversity of TorontoTorontoCanada
  2. 2.Department of Biochemistry and Structural BiologyHospital for Sick ChildrenTorontoCanada
  3. 3.Protein Engineering Network Centres of Excellence and Departments of Medical Genetics and Microbiology, Biochemistry and ChemistryUniversity of TorontoTorontoCanada

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