Preparation Of Immobilized Metal Affinity Magnetic Nanoparticles And Its Application To Purification Of His₆-tagged Protein

Immobilized metal affinity magnetic nanoparticles (IMAN), a new technology for purification of His6-tagged protein, is being researched on the principle of immobilized metal affinity chromatography (IMAC). IMAN works fast and effectively not only because of the high affinity between transition metal ions Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺ and the imidazolyl, thiol group, and indyl of histidine, cysteine, and tryptophane, but also because of its magnetic response and nano size which make purification process easier and adsorption capacity higher. There are two parts in this article:1. preparation and characterization of IMANThe IMAN is prepared by carboxyl-group functionalized magnetic nanoparticles as support, IDA as chelating legend and transition metal ions Cu²⁺, Ni²⁺, Zn²⁺ or Co²⁺ as chelating center ions. IDA is coupled to the carboxyl group by EDC and NHS.The TEM images show that there's no diffirences between nanoparticles with IDA and those without IDA. The FTIR spectra of nanoparticles indicates that IDA is coupled and transition metal ions Cu²⁺, Ni²⁺, Zn²⁺ and Co²⁺ are chelated on the IMAN respectively. The detection by FAAS also shows that metal ions are adsorbed by the nanoparticles with or without IDA but the reason is unknown.2. purification of His6-tagged proteinThe IMAN is used to purify His6-tagged protein. The protein purity and concentration are researched by SDS-PAGE and UV method. The results are as follows:(1) There are several influencing factors to adsorption and elution. Binding time effects adsorption quantity and 45 min is the best one for adsorption quantity; the pH of binding Tris buffer (7.1-8.9) has a great effect on adsorption quantity: more His6-tagged protein is adsorbed when the pH is low (7.1-7.4) and when the pH is higher than 8.6, there's little adsorbed; the concentration of imidazole eluting buffer influences greatly on elution: the imidazole about 0.8 mol/L elutes more His6-tagged protein than those eluted by imidazole above or below 0.8 mol/L.(2) Transition metal ions Cu²⁺, Ni²⁺, Zn²⁺ and Co²⁺ as chelating center ions have different abilities on binding and eluting. Compare the protein quantities and get the following results: for adsorption quantity of target protein, Cu²⁺≈Ni²⁺＞Zn²⁺＞＞Co²⁺; for elution, Zn²⁺＞Ni²⁺＞Cu²⁺＞＞Co²⁺; for remaining after eluting, Cu²⁺≈Ni²⁺＞Zn²⁺＞Co²⁺. It has a relation to the stability of the coordination compound. Some protein is adsorbed and eluted in the control groups (no metal ions and no IDA or metal ions), even more than the one using Co²⁺-IMAN. In a word, Zn²⁺-IMAN is better for the purification.(3) L₄ (2³) orthogonal experiment is done (metal ions: Cu²⁺, Zn²⁺; the concentration of imidazole eluting buffer: 0.5, lmol/L; the pH of binding Tris buffer: 7.1, 8.0) and the best choice is that Zn²⁺, Tris buffer at pH 7.1 when binding, 0.8 mol/L imidazole when eluting.