Ni nta agarose

To purify KlLeu4 protein (KLLA0F23529, syntenic), supernatant was also purified through an equilibrated nickel column (Ni-NTA Agarose, Quiagen), and following the protocol of purification of KlLeu4 with the difference that 2 extra wash steps were added. The first extra wash was 2 volumes of 30 mmol L^–1 imidazol and the second was 2 volumes of 60 mmol L^–1 imidazol. To purify KlLeu4BIS protein (KLLA0D14201, non-syntenic), the supernatant was loaded on an equilibrated nickel column (Ni-NTA Agarose, Quiagen), washed with 100 volumes of lysis buffer, 50 volumes of 2 mmol L^–1 imidazol, 2 volumes of 5 mmol L^–1 imidazol, 2 volumes of 10 mmol L^–1 imidazol and 2 volumes of 20 mmol L^–1 imidazol. The protein was eluted with 2 volumes of 50, 100, 200, and 300 mmol L^–1 imidazol and stored at 4^oC until used. KlLeu4 and KlLeu4BIS homogeneity was verified by denaturing with a polyacrylamide gel electrophoresis (12%, SDS-PAGE) and the gel stained with Coomassie Blue. Proteins were 10-fold concentrated with AmiconR Ultra-15 10K Centrifugal Filter Devices (Millipore), and then diluted to the original sample volume with assay buffer (50 mM K₂HPO₄, pH 7.5) three “washing out” cycles were performed.

cDNA encoding the Arabidopsis NDF5 without its chloroplast transit peptide predicted by ChloroP 1.1^{62 (link)} was amplified. The amplified sequence was digested with AseI and XhoI and cloned into NdeI and XhoI digested pET-22b(+) (Novagen) using Ligation high (Toyobo). Expression of the recombinant proteins was induced by 1 mM isopropyl β-D-thiogalactopyranoside at 37 °C for 4 h in host Escherichia coli strain Rosetta (DE3) pLysS cells (Novagen). After induction, the cells were harvested in 20 mM potassium phosphate buffer (pH 7.4) containing 40 mM imidazole, 500 mM NaCl, and cOmplete™ EDTA-free protease inhibitor cocktail (Roche). The inclusion bodies were pelleted from sonicated cells at 3000g for 15 min and solubilized in 20 mM potassium phosphate buffer (pH 7.4) containing 40 mM imidazole, 500 mM NaCl, and 6 M guanidine hydrochloride. Insoluble material was removed by centrifugation at 48,000g for 1 h. The supernatant was incubated with Ni-NTA Agarose (Qiagen) for 1 h. The Ni-NTA Agarose was washed with 20 mM potassium phosphate buffer (pH 7.4) containing 40 mM imidazole, 500 mM NaCl, and 4 M urea. The recombinant proteins were eluted with 20 mM potassium phosphate buffer (pH 7.4) containing 500 mM imidazole, 500 mM NaCl, and 4 M urea. Polyclonal antisera were raised against the purified recombinant protein in a mouse (T. K. Craft, Maebashi, Japan).