Preparation And Application Of A Novel Ph-Sensitive Metal-Chelate Affinity Precipitant

In the production process of biopharmaceutical proteins, most of target proteins were often expressed with a 6×Histag using genetic engineering techniques, which is convenient to be purified by immobilized metal ion affinity chromatography (IMAC). However, in IMAC, some severe problems concerning column operation are encountered: complicated operation, high cost, difficulty in scaling up. Therefore, development of a simple and efficient purification protocol for the 6×His-tagproteins has always been a challenging task for scientists.Affinity precipitation is a novel purification protocol of proteins with such advantages as simple operation, low cost and easy amplification. In this paper, we developed a novel pH-sensitive metal-chelate affinity precipitant ES-IA-Ni²⁺, using pH-sensitive polymer Eudragit S-100 with a covalently linked ligand loaded with Ni²⁺. Besides, using myoglobin (Mb) as a model protein, purification effect of ES-IA-Ni²⁺ for Mb was systematically studied.The results showed that ES-IA-Ni²⁺ has many advantage, such as fast response, good precipitation behavior and sensitive conditions. In addition, the Ni²⁺ of ES-IA-Ni²⁺ is so stable that the leakage rate is less than one ten thousandth after being reused. 60 minitutes later, Mb was completely absorbed by coordination of ES-IA-Ni²⁺ and formed complex Mb-ES-IA-Ni²⁺. The adsorption capacity of ES-IA-Ni²⁺ to Mb is about 0.7 mg Mb/g ES-IA-Ni²⁺. When changing the pH, Mb- ES-IA-Ni²⁺ was completely precipitated from the solution. Compared with Lysozyme (Lys), ES-IA-Ni²⁺ could absorb Mb which has more His residue through coordination with yield of 95%, while the Lys containing less His residues couldn't be absorbed by ES-IA-Ni²⁺ through coordination.These results reported here suggested that the novel pH-sensitive metal-chelate affinity precipitant ES- IA-Ni ²⁺ may be used in the purification of proteins in future. 6×His-tag...