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