Isotopic Labeling of Proteins in M9 Media

Isotope labeling of proteins was achieved by expressing protein in 2×M9 minimal media (28 (link)) with the relevant isotopes supplemented to the media (H₂O supplemented with (¹⁵NH₄)Cl for [U-¹⁵N]-labeled protein or with both (¹⁵NH₄)Cl and d-(¹³C)-glucose for uniform double labeling [U-¹³C,¹⁵N], or D₂O supplemented with (¹⁵NH₄)Cl yielding [U-²H,¹⁵N]-labeled proteins or D-(²H,¹³C)-glucose for uniform triple labeling [U-²H,¹³C,¹⁵N]). For specific methyl group labeling, Met, Ala, Leu, Val and Ile (MALVI^proS)-labeled DinB sample was produced in Bioexpress rich (2%) D₂O-based 2× M9 media supplemented with (¹⁵NH₄)Cl, d-(²H, ¹²C)-glucose as well as 50 mg/l 2-Ketobutyric acid-4–¹³C,3,3-d₂ sodium salt hydrate (Isoleucine), 4 vials/l DLAM-LV^proS-kit (2-(¹³C)-methyl-4-(D₃)-acetolactate (valine/leucine proS methyl group only), 50 mg/l [U-²H, ¹³CH₃] methionine, and 50 mg/l 2-[²H], 3-[¹³C] L-alanine (alanine) precursors added 1 h prior to induction (29–31 (link)). Bioexpress, methionine, and alanine were purchased from Cambridge Isotopes Laboratories and the DLAM-LV^proS precursors from NMR-Bio. All other isotopes were purchased from Merck.