The Study Of 6×His-tagged Fusion Protein Purification

6×His is a common tag in purification and detection of fusion protein. Ni-NTA (Nitrilotriacetic acid) and Ni-IDA(Iminodiacetic acid) chelating resin have been used to isolate 6×His-tagged fusion proteins based on the interaction between histidine tag and Ni²⁺ in chelating resin. Recently, carboxymethylated aspartate(CM-Asp) bound with Co²⁺ has been proved to exhibit high selectivity and less metal leakage during the purification of 6×His-tagged fusion proteins. Forthermore, the purification of 6×His-tagged fusion proteins using anti-6×His antibody has attracted the interests of scientists in this field. Herein, the purification methods of 6×His-tagged fusion proteins based on CM-Asp chelating medium loaded with Co²⁺ and the anti-6×His antibody are described in detail.Using Sepharose CL-6B as support, 3-Chloro-l,2-epoxypropane as activated agent, carboxymethylated aspartate (CM-Asp) as chelating ligand, A chelate affinity chromatographic medium based on Co²⁺(Co-CM-Asp-Sepharose) was prepared and used to purify 6×His-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200μL of lysate, the incubation time, wash condition and the imidazole concentration in the elution buffer were optimized. The results show that 60μL of Co-CM-Asp-Sepharose (50% suspension) was suitable for the protein purification from 200μL of lysate, the optimal incubation time of medium and lysate was 30 min, and the optimal imidazole concentration in the eluting buffer was 200 mmol/L. In a big 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 (product of Qiagen), the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the protein possesses higher purity.6×His was conjugated with KLH(Keyhole limpet hemocyanin) using EDC(1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride) as a cross-linker and the conjugation was used as immunogen to immunize rabbits, antiserum raised was purified by the precipitation with saturated ammonium sulfate. The anti-6×His polyclonal antibody was immobilized on the surface of magnetic particles via covantly interaction and used to purify 6×His-tagged fusion protein CD155D1 based on immunoaffinity chromatography. The results show that the anti-6×His polyconal antibody can be used to purify 6×His-tagged fusion protein. However, the method need further optimization. One of the influencing factors is the large amount of anti-KLH antibody existing in anti-6×His polyclonal antibody. Therefore, using GoldMag coated with KLH, the anti-KLH antibody was removed from the polyclonal antibody, the anti-6×His antibody in supernatant after magnetic separation will be used in the 6×His-tagged fusion protein purification in the next steps.