Superdex 75 hiload 26 600

The auxiliary enzyme tryptophan oxidase (VioA) from Chromobacterium violaceum was expressed in BL21 Gold (DE3).^[24c] Transformed E. coli strains were grown in 4L LB medium supplemented with kanamycin at 37 °C to an OD₆₀₀ of 0.6. Then, protein expression was induced with 0.5 mm IPTG and cells were incubated overnight at 20 °C. Cells were harvested by centrifugation and the pellets were resuspended in 20 mm Tris⋅HCl pH 8.0, 300 mm NaCl, and 20 mm imidazole. The target protein was obtained from the supernatant after sonication and repeated centrifugation steps. VioA was captured by nickel‐affinity chromatography (HisTrap FF Crude column, 5 mL, GE Healthcare, Chicago, IL, USA) and eluted with a linear gradient of imidazole (10→500 mm). Fractions containing the target protein were identified by SDS‐PAGE analysis, pooled, and further purified by size‐exclusion chromatography (Superdex 75 HiLoad 26/600, GE Healthcare, Chicago, IL, USA) with 20 mm Tris⋅HCl pH 8.0 as running buffer. Fractions containing the purified protein were pooled and dripped into liquid nitrogen for storage at −80 °C.

The genes coding for TrpA and TrpB were heterologously expressed in BL21 (DE3) Rosetta and BL21 Gold (DE3), respectively. Cells containing expression vectors with the respective genes were grown at 37 °C to an OD₆₀₀ of 0.6 in 2–6 L lysogeny broth (LB) medium supplemented with kanamycin. At this point, 0.5 mm isopropyl‐β‐d‐thiogalactopyranosid (IPTG) was added to induce gene expression and cells were incubated overnight at 20 °C. The cells were harvested by centrifugation, resuspended in 100 mm potassium phosphate (KP) pH 7.5, 300 mm KCl, 20 mm imidazole (20 mL per 1 L cell suspension), and disrupted by sonication. Cell debris and insoluble aggregates were removed by centrifugation. Proteins were purified from the supernatant by nickel‐affinity chromatography (HisTrap FF Crude column, 5 mL, GE Healthcare) with a linear gradient of imidazole (20 mm→500 mm) followed by size‐exclusion chromatography (Superdex 75 HiLoad 26/600, GE Healthcare, Chicago, IL, USA) using 100 mm KP pH 7.5, 300 mm KCl as buffer. Fractions containing the purified protein were pooled and dripped into liquid nitrogen for storage at −80 °C.