Preparation Of Cobalt Chelate Affinity Mediums And Their Application In Purification Of 6×His-tagged Proteins

Immobilized metal ion affinity chromatography (MAC) or metal chelate affinity chromatography (MCAC) is one of the important methods used in the separation of protein due to its high selectivity and simplicity. IMAC uses covalently bound chelating compounds in solid chromatographic methods to aid in entrapping transition-metal ions Ni²⁺, Co²⁺, which then serve as affinity ligands for 6×His-tagged fusion proteins, making use of coordinative binding of 6×His exposed on the protein surface. As a result, this method has gained broad popularity in resent years. Furthermore, the purification of 6×His-tagged fusion proteins using magnetic beads has attracted the interests of scientists throughout this field. Therefore, using polymer micrsphere (Agarose CL-6B) and magnetic particle as carriers, the metal chelate affinity chromatography purification methods of 6×His-tagged fusion proteins based on carboxymethylated aspartate (CM-Asp) chelating resin and magnetic beads loaded with Co²⁺ are described as the following:1. Using AgaroseCL-6B as a support, 3-Chloro-l,2-epoxypropane as a activated agent, carboxymethylated aspartate (CM-Asp) as a chelating ligand, A chelate affinity chromatographic medium based on Co²⁺(Agarose-CM-Asp-Co) was prepared and the large-scale purification system of 6×His-tagged proteins was established under both denatured and native conditions. The efficiency for purification of 6×His-tagged protein by Agarose-CM-Asp-Co was then compared with Agarose-CM-Asp-Ni and commercial Ni-NTA-Agarose (Qiagen). The results show that the optimal binding time is 15 min and the binding capacity of DXS by Agarose-CM-Asp-Co is 14.8 mg/mL under native condition. For the denatured condition, the optimal binding time is 20 min and the binding capacity of CD155D1 achieves 7.6 mg/mL. Compared with nickle chelate affinity chromatographic mediums, Agarose-CM-Asp-Co possesses higher selectivity and specificity. 2. Superparamagnetic silica nanoparticles with immobilized metal affinity ligand (CM-Asp-Co), named Affimag-CM-Asp-Co, were prepared and used for the purification of 6×His-tagged fusion proteins in small-scale samples. The amount of Affimag-CM-Asp-Co reacted with 500μL of lysate, the incubation time and the buffer system were optimized. The results show that 1 mg of Affimag-CM-Asp-Co is suitable for the protein purification from 500μL of lysate, the optimal incubation time of medium and lysate is 10 min and the nanoparticles has high adsorption capacity for DXS(9.4μg/mg) under native conditions. Compared with metal chelate affinity agarose, Affimag-CM-Asp-Co has less procedural steps, shorter separation time and prior selectivity in relation to the purification of 6×His-tagged proteins.