Purification of Histidine-Tagged Recombinant Protein

rhAA was purified as histidine-tagged (His-tag) fusion protein from rice cell culture medium using affinity chromatography on a Ni²⁺—NTA agarose (Qiagen, Hilden, Germany) column, following the manufacturer’s instructions. Briefly, 50 mL of a 7-day induced culture medium was centrifuged at 13,200 rpm at 4 °C for 20 min and filtrated through a 0.2 µm filter to remove debris. The column was washed out of the preservative, and 700 µL of Ni²⁺—NTA agarose resin was added into the open column. After equilibrating with 5 mL of 10 mM Imidazole, the prepared culture medium (added 1M Imidazole to the culture medium to obtain the final 10 mM) was loaded to the purification column at a flow rate of about 0.5 mL/min. The Ni²⁺—NTA agarose column was washed with 10 mL of 10 mM Imidazole. Binding proteins were eluted using 250 mM Imidazole in a total volume of 1 mL. Fractions were collected, and purity was visually assessed by SDS-PAGE and Western blot analysis.