Synthesis Of Hydroxyapatite Nanoparticles And Their Purification Of His-tagged Proteins

In this paper, nano-sized HAP/IDA-Ni2+, Fe3O4/HAP-Ni2+, Ni(OH)2/HAP and Ni(OH)2/HAP-RBH were used as affinity adsorbents to separate and purify histidine-tagged protein, using the affinity interaction between Ni2+ and His-tagged proteins. Their morphology, structure and binding protein ability were characterized by a variety of modern analytical methods. The main research contents and conclusions of this paper are as follows:(1) Preparation of HAP/IDA-Ni2+ NPs for His-tagged protein separationhydroxyapatite(HAP) nanoparticles(NPs) were prepared by hydrothermal method in a simulated body fluid(SBF). The HAP NPs have an average diameter of 50 nm and present porous structure. By uselizing surface hydroxyl groups, the HAP NPs are further modified with IDA, followed by chelating Ni2+ ions. The HAP/IDA-Ni2+ NPs as novel adsorbent can capture directly His-tagged proteins from the mixture of lysed cells. Results indicated that these NPs present negligible nonspecific adsorption and high protein binding ability, and their specificity and affinity toward His-tagged proteins can remain after 4 times of recycling.(2) Preparation of Fe3O4/HAP-Ni2+ NPs for His-tagged protein separationFe3O4/HAP NPs were synthesized by hydrothermal method. The obtained NPs have excellent magnetic responsibility and can immobilize metal ions. After chelating Ni2+ ions, Fe3O4/HAP-Ni2+ NPs can be used to enrich His-tagged proteins directly from the mixture of lysed cells. Results show that Fe3O4/HAP-Ni2+ NPs present negligible nonspecific protein adsorption and high protein binding activity with the saturation capacity being 12.98 mmol/g.(3) Preparation of Ni(OH)2/HAP NPs for His-tagged protein separationNickel hydroxide/hydroxyapatite(designated as Ni(OH)2/HAP) NPs have been designed and synthesized by hydrothermal method. The size and morphology of Ni(OH)2/HAP sample depend strongly on the reaction condition, and introduction of small amount of Ni2+ ions would be propitious to get the smaller size NPs. By taking advantage of the high affinity of Ni2+ ions toward His-tagged proteins, the Ni(OH)2/HAP NPs can be used to enrich and separate His-tagged proteins directly from a mixture of lysed cells, and their universality was evaluated by separating three kinds of His-tagged proteins. The Ni(OH)2/HAP NPs present negligible nonspecific protein adsorption and high protein binding ability.(4) Preparation of Ni(OH)2/HAP-RBH NPs for His-tagged protein separationNi(OH)2/HAP NPs coated with rhodamine B hydrazide(RBH) were successfully synthesized in three steps. First, Ni(OH)2/HAP NPs were synthesized by a facile hydrothermal route in the presence of citric acid. Second, rhodamine B was reacted with hydrazine hydrate to obtain RBH. Finally, RBH was conjugated on the surface of Ni(OH)2/HAP NPs to form Ni(OH)2/HAP-RBH. As affinity adsorbents, these NPs can directly purify His-tagged proteins from a mixture of lysed cells, and exhibit high protein adsorption capacity with a maximum value of 115 mg/g. A prominent fluorescence enhancement at 578 nm was observed in the presence of Pt2+, accompanied by a change in absorption spectrum. These NPs display excellent selectivity and sensitive recognition of Pt2+ in comparison with other metal ions and anions with a detection limit of 3.0×10-8 mol/L. These NPs exhibit low cytotoxicity and can be used as a probe for Pt2+ and living cell imaging.