Chemicals and reagents were purchased from commercial sources and used without further purification. All cultures were grown in Terrific Broth supplemented with 100 μg/mL carbenicillin (TBcarb). Cultures were shaken in a New Brunswick Innova 4000 (shaking diameter 19 mm), with the exception of the 96-well deep-well plates (USA Scientific), which were shaken in a Multitron INFORS HT (shaking diameter 50 mm). Lysis buffer was composed of 50 mM potassium phosphate, pH 8.0 (KPi buffer), supplemented with 100 or 200 µM pyridoxal 5’-phosphate (PLP). Heat lysis was performed in a 75 °C water bath (Fisher) for >1 h. Protein concentrations were determined using a Pierce™ BCA Protein Assay Kit (Thermo Scientific). Reactions were performed in KPi buffer. Liquid chromatography/mass spectrometry (LCMS) was performed on an Agilent 1290 UPLC-LCMS equipped with a C-18 silica column (1.8 μm, 2.1 × 50 mm) using CH3CN/H2O (0.1% acetic acid by volume): 5–95% CH3CN over 2 min; 1 mL/min. Liquid chromatography/mass spectrometry (LCMS) was also performed on an Agilent 1260 HPLC-MS equipped with Agilent InfinityLab Poroshell 120 EC-C18 column (2.7 μm, 4.6 × 50 mm): hold 5% CH3CN for 0.5 min, 5–95% CH3CN over 2 min; 1 mL/min.
TrpB variants selected for further characterization were cloned into a pET-22b(+) vector with a C-terminal 6X His-tag and transformed into E. coli BL21(DE3) cells (Lucigen).
TrpB variants selected for further characterization were cloned into a pET-22b(+) vector with a C-terminal 6X His-tag and transformed into E. coli BL21(DE3) cells (Lucigen).