A cDNA fragment encoding CsMRP4-NBD1 was amplified using PCR (Additional file 1: Table S1). The purified PCR product was then subcloned into pET23b and confirmed by colony PCR and restriction enzyme digestion. Plasmid DNA of the positive clone was extracted using the QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) and sequenced (Macrogen, Seoul, Korea). The correct construct was then transformed in Escherichia coli BL21[DE3]pLysS (Novagen, San Diego, CA, USA) by heat-shock at 42 °C for 30 s and spread on LB/ampicillin/chloramphenicol agar. After overnight incubation at 37 °C, a single colony was picked from the LB plate and grown in LB/ampicillin liquid medium by shaking vigorously at 37 °C. The recombinant(r) CsMRP4-NBD1 was then induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (TaKaRa, Shiga, Japan) for 5 h. The bacteria were then harvested, and recombinant protein was purified as described previously .