| In recent years, with the development of techniques for geneticengineering, the techniques in separation recombination proteins become more andmore important. It is a hard and heavy task to isolate and purify proteins from thecomplicated expressed system. Based upon the difference in size, shape, charge,molecular weight and specific binding affinity of proteins, the methods for proteinseparation have been set up. Immobilized metal ion affinity chromatography (IMAC,also named metal chelate affinity chromatography) is one of the important methodsextensively used in separation of protein due to its high selectivity and simplicity.Researches indicate that there are many influence factors in the protein purificationprocess using IMAC, such as pH, the concentration of the salt, the way of elution, etc.However, the component of matrix, the ligand and the kinds of metal ions attached tothe matrix are the key factors. Agarose has some superior performance in hydrophilicity,stability, and nonspecific adsorption with soluble proteins. As support, Agarose can benot only used in normal condition, but also in denaturalization (for example, 6 mol/Lhydrochloric acid carbamidine or 8 mol/L urea). Herein, the preparation of IMACmatrixes were conducted using agarose as support, carboxymethylated aspartate(CM-Asp) as chelating ligand and Co2+ and Ni2+ as chelating center ions. The mainworks include the following parts:1. IMAC matrixes named Agarose-CM-Asp-Co and Agarose-CM-Asp-Ni wereprepared using agarose as support, 3-Chloro-1,2-epoxy -propane as activated agent,carboxymethylated aspartate (CM-Asp) as chelating ligand, the Co2+ and Ni2+ as centerions. IR spectra of agarose, Agarose-CM-Asp-Co and Agarose-CM-Asp-Ni indicate thatCo2+ and Ni2+ were successfully chelated on the support respectively. SEM images ofagarose and Agarose-CM-Asp-Ni show that both kinds of microspheres are intact insurface. This feature of morphology is useful to reduce the nonspecific adsorption.Based on this IMAC matrixes, the purification of 6×His-tagged protein were conductedand the SDS-PAGE analysis to elution of target protein were also investigated. The results show that the matrix bonded with Co2+ is better than with Ni2+ in purification ofthe protein. Additionally, Agarose-CM-Asp-Co has high selectivity and low nonspecificabsoption.2. A series of mediums including Agarose-0.5CM-Asp,Agarose-CM-Asp andAgarose-2CM-Asp were prepared employing SepharoseC1-6B as support,3-Chloro-1,2-epoxypropane as activated agent, different amount of carboxymethylatedaspartate (CM-Asp) as chelating ligands. The amount of carboxyl goups activated in theprepared mediums is 3.34×10-5, 3.83×10-5 and 4.14×10-5 mol/L, respectively. The resultindicates that the Agarose are saturated combined with CM-Asp in the given conditions.The amount of metal ion in Agarose-CM-Asp-M was determined by atoms absorptionspectra. The results testify the conclusion mentioned above. Agarose-CM-Asp-Mmediums with different amount of metal ions were used to purify 6×His-tagged proteinsand the results illustrate that the amount of metal ions in the mediums only influencesthe amount of chelated protein, but not the purity.3. The conditions for purification of 6×His-tagged protein including amount ofAgarose-CM-Asp-Co, the binding time between Agarose-CM-Asp-Co and lysate, thewashing condition and the imidazole concentration in the eluting buffer were optimized.The results show that 60μL (v/v 50% suspension) of Agarose-CM-Asp-Co solution issuitable for purification of the dissolved protein from 200μL of lysate, the optimalbinding time between Agarose-CM-Asp-Co and lysate is 30 min, and the optimalimidazole concentration in the eluting buffer is 200 mM. The efficiencies forpurification of 6×His-tagged proteins by Agarose-CM-Asp-Co and Ni-NTA-Agarosemade by Qiagen were compared. Compared with commercialized mediumNi-NTA-Agarose, the Agarose-CM-Asp-Co has prior selectivity and quality inpurification of 6×His-tagged proteins.
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