Heterologous Expression of FhDFR Proteins in Escherichia coli and In vitro Enzyme Assay

Heterologous expression of FhDFR genes, which were determined to restore the phenotype of Arabidopsis mutant tt3-1, and enzyme assay was carried out following the previously described methods (Sun et al., 2015, 2016). Briefly, FhDFR genes were subcloned into the pET28a vector and expressed as N-terminal His-tagged proteins. An empty vector and vectors harboring different FhDFR cDNAs were used for transformation of E. coli strain BL21 (DE3). Then the transformants were pre-cultured at 37°C overnight in LB media containing 50 mg L^-1 kanamycin. The preculture was then transferred to fresh LB media containing 50 mg L^-1 kanamycin and cultured at 37°C until an A₆₀₀ of 0.6 was reached. Recombinant proteins were then induced by adding 0.2 mM isopropyl-b-d-thiogalactopyranoside (IPTG), and the optimal induction condition was 28 h and 15°C for FhDFR1 and FhDFR2, 28 h and 20°C for FhDFR3, respectively. After induction, the cells were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS, pH 7.4), and disrupted by sonication. After centrifugation at 13,225 g for 20 min, the supernatant containing crude proteins was then applied to 3 ml PBS-equilibrated Ni Sepharose column (GE Healthcare). The column was then washed to remove non-specifically bound proteins using gradient imidazole in PBS. The purified proteins were eluted from the column using 100 mM imidazole in PBS. Eluted FhDFR proteins were desalted in PBS to remove the imidazole at 4°C. The desalted FhDFR proteins were then concentrated and assessed by SDS-PAGE with Coomassie Brilliant Blue staining (Supplementary Figure S1). After that, their concentrations were detected by NanoDrop 1000 (Thermo scientific) Spectrophotometer before enzymatic assays.

Substrate specificities of FhDFR proteins were carried out as described by Cheng et al. (2013). Shortly, DHK, DHM, and DHQ bought from Sigma were dissolved in methanol at 10 mg/mL. A 500 μl reaction mixture consisting of 370 μL of 100 mM Tris-HCl buffer (pH7.0), 70 μL of 0.5 mg/ml FhDFR enzyme extract, 10 μL of substrate, and 50 μL of 20 mM NADPH was kept at 30°C for 30 min. 20 μL of reaction solution was resolved on an ACCHROM XUnion C18 column. The column was eluted with solvent systems A (1% H₃PO₄ in water) and B (methanol) under the following conditions: 0 min, 15% B; 0–20 min, 15–60% B; 20–30min, 60–15% B. Detection was monitored at 280 nm, the maximum absorbance wavelength for most of the substrates and products.