Ni nta pre packed column

The full-length CDS of Rd3GT1 and Rd3GT6 was introduced into the EcoR I/EcoR I and BamH I/Hind III site of pET32a (+) expression vector, respectively, and transformed into E. coli strain BL21 (DE3). On the second day, transformed E. coli expressing the recombinant Rd3GTs protein was cultured in liquid Luria-Bertani (LB) medium at 37°C till the value of OD₆₀₀ of 0.6 was reached; after that, the cultures were induced several times for 36 and 48 h at 15°C through adding 0.2 mM of isopropyl-β-d-thiogalactopyranoside (IPTG). The cells were then collected by centrifugation (5,000 rpm, 10 min, 4°C) and suspended in phosphate-buffered saline without protein inhibitor (PBS, pH 7.4). After sonication in ice, the cell debris was removed and filtered with a 0.45 μm filter (Millipore). Next, the supernatant which contained soluble recombinant protein was purified via Ni-NTA pre-packed column (TransGen, China) and its purity was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of recombinant Rd3GTs protein was measured by NanoDrop 1,000 Spectrophotometer (Thermo scientific, Waltham, MA, USA).

The open reading frame of OjCHI was amplified using the primers (OjCHIF3 and OjCHIR3) in Supplementary Table S1 and subcloned into the pET-32a expression vector. After verification by sequencing, the recombinant construct as well as the empty vector were transformed into Escherichia coli strain BL21, respectively. The overnight bacterial cultures obtained from a single transgenic colony were diluted into LB medium and grown to OD₆₀₀ = 0.6, at which point 0.35 mM isopropyl β-d-thiogalactoside was added to induce recombinant protein expression at 30°C for 10 h. Then the cells were harvested through centrifugation at 6,000 rpm for 10 min and disrupted by sonication on ice. The His₆-tagged recombinant proteins were purified using Ni-NTA pre-packed column (TransGen, China) following the manufacturer’s recommendations and its purity was finally tested by SDS-PAGE.