Preparation of Metal Chelate Affinity Chromatographic Medium and Its Application in the Purification of 6× Histidine-tagged Protein

Using Sepharose CL-6B as support, 3-Chloro-1,2-epoxypropane as activated agent and carboxymethylated aspartate (CM-Asp) as chelating ligand, a chelate affinity chromatographic medium based on Co2+, named Co-CM-Asp-Sepharose, was prepared and used to purify 6×His-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200 μL of lysate, the incubation time, wash condition, and the imidazole concentration in the elution buffer were optimized. The purification results with Co-CM-Asp-Sepharose and commercialized Ni-NTA-Agarose were compared. The CD155D1 fusion protein was also purified from 5∼mL of lysate and the amount of protein was determined by Bradford method. The results show that 60 μL of Co-CM-Asp-Sepharose (50% suspension) was suitable for protein purification from 200 μL of lysate, the optimal incubation time of medium and lysate was 30∼min, the optimal imidazole concentration in the eluting buffer was 200∼mmol/L, and 200 μg of fusion protein was obtained. In a large-scale experiment, 4.6∼mg of fusion protein was obtained from 5∼mL of lysate using 1.5∼mL of Co-CM-Asp-Sepharose (50% suspension). Compared with Ni-NTA-Agarose, the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the purified protein possesses higher purity.