Fig. 3 in Genome-wide identification and characterization of genes involved in melatonin biosynthesis in Morus notabilis (wild mulberry)

Fig. 3. Purification and enzymatic analyses of recombinant His-tagged MnTDC and recombinant His-tagged MnT5H2. A: Purification of recombinant His-tagged MnTDC (lane1: Protein Marker; lane2: pCold TF (+IPTG); lane3: recombinant protein (-IPTG); lane4: recombinant protein (+IPTG); lane5: purified protein). B: Mass spectrum of compounds detected using UPLC-MS/MS. a: tryptamine standard; b: Chromatogram of tryptophan catalyzed by MnTDC. C: Determination of Km and V of MnTDC for tryptophan. The MnTDC (1.0 μg) was co-incubated with different concentrations of tryptophan for 20 min at 37 ◦C. Trp: tryptophan; Try: max tryptamine. D: Purification of recombinant His-tagged MnT5H2 (lane1: Protein Marker; lane2: pCold TF (+IPTG); lane3: recombinant protein (-IPTG) lane4: recombinant protein (+IPTG); lane5: purified protein). E: Mass spectrum of compounds detected using UPLC-MS/MS. a: serotonin standard; b: Chromatogram of tryptamine catalyzed by MnT5H2. F: Determination of Km and Vmax of MnT5H2 for tryptamine. The MnT5H2 (1.0 μg) was co-incubated with different concentrations of tryptamine for 20 min at 28 ◦C. Ser: serotonin. " IPTG" stand without addition of 1 mM IPTG to the E. coli BL21 (DE3) culture, "+IPTG" stand addition of 1 mM IPTG to the E. coli BL21 (DE3) culture. "TF" is Trigger Factor, a prokaryotic ribosome-related molecular chaperone that facilitates the translation and folding of peptides. "*" indicates Trigger factor.