Preparation Of Immobilized Metal Affinity Sepharose CL-6B And Its Application To Purification Of His₆-tagged Protein

Immobilized metal Ion affinity chromatography is a good way for the purification of protein.Among them,immobilized metal ion chelate Sepharose CL-6B,a new technology which is used for the purification of His6-Tagged protein,has attracted much attention in recent years.The method mainly uses ligands which are fixed on the surface of microspheres,chelating metal ion to form chelate compound by covalent interaction,then the ligands on the surface of protein attach with chelate compound through the coordination effect between them.The specific research contents are as follows:1.Two chelate affinity chromatographic mediums (SepharoseCL-6B-IDA-Ni2+ and Sepharose-CL-6B-CM-ASP-Co2+) are prepared using SepharoseCL-6B as the carrier, epichlorohydrin as the activating agent, the iminodiacetic acid and carboxymethylated aspartic acid as chelating ligands. It proves by SEM:the appearance of SepharoseCL-6B has not changed after activation,and the agarose is not broken in the process of activation.By exploring the influence factors of Sepharose CL-6B-IDA-Ni2+,the study finds:while the ratio between the epoxychloropropane and the matrix is 0.5, the ratio between the iminodiacetic acid and the matrix is 2,and reaction time is 1h,the effciency of activation is best.2.They purify small scale His6-Tagged protein using Sepharose-CL-6B-IDA-Ni2+ and Sepharose-CL-6B-CM-ASP-Co2+.The effects of them are compared with commercial products (Ni-NTA medium).Through the analysis of the SDS-PAGE:the best effect of the purification of the protein is Ni-IDA medium,which is better than CM-ASP-Co medium and the commercial product (Ni-NTA medium),and the purified target protein almost has not any impurities with Ni-IDA medium,and the high-purity protein can be obtained by one step of the purification.3.The chelating ligand-N-[5-amino-l-carboxypentyl]-iminodiacetic acid is synthesized.The stucture of the product is determined by IR and NMR.The purification of the protein will be further studied using N-[5-amino-1-carboxypentyl]-iminodiacetic acid as a chelating ligand.The medium-SepharoseCL-6B-IDA-Ni2+ can be commercialized and used for the purification of large-scale histidine protein.