Synthesis Of Silica-based Nano-aborbent And Their Affinity Separation Of His-tagged Proteins

In this paper, micro/nanosized SiO2-IDA-Ni2+, NiSiO3, Fe3O4@NixSiOyand NiO were used as affinityadsorbents to separate and purify protein tagged with hexahistidine (denoted as His-tagged proteins), usingthe affinity interaction between Ni2+and hexahistidine. Their morphology, structure and binding proteinability were characterized by a variety of modern analytical methods. The main research contents andconclusions of this paper are as follow:(1) Preparation and biofunction of SiO2nanospheres for His-tagged protein separationSiO2nanospheres with an average diameter of200nm was first synthesized by hydrolysis of TEOS.Then these nanospheres were further modified to form SiO2-IDA nanospheres by conjugating epoxy silanecoupling agent and chelating ligand. After adsorbing Ni2+, SiO2-IDA-Ni2+nanospheres was formed and wasused for purifying of His-tagged fusion protein. SEM, FTIR, TG and BET were used to characterize thesurface morphology, structure and composition of the nanospheres. The separated His-tagged proteins weredetected with SDS-PAGE and the quantitative analysis was carried by BCA protein quantitative kit. Resultsindicated that the SiO2-IDA-Ni2+nanospheres had a good ability to separate His-tagged protein, with anadsorption capacity of28.3mg/g.(2) Preparation of hollow nickel silicate nanospheres for His-tagged protein separationHollow nickel silicate nanospheres were synthesized by the method of hydrothermal with SiO2nanospheres as template. Their morphology, structure and properties were characterized by using SEM,HRTEM, XRD and XPS. There exsist many Ni2+sites inside and outside the surface of nanospheres, so they could be directly used for separation and purification of His-tagged protein. The results showed thatthe hollow NiSiO3nanospheres showed good specificity and separation effeciency in purification of protein.And after reused for many times, it still kept an effective separation effect and regeneration.(3) Preparation of Fe3O4@NixSiOymicrospheres for His-tagged protein separationAfter synthesizing of the magnetic Fe3O4microspheres via a solvothermal route, a layer of SiO2shellwas coated by sol-gel method on the surface of Fe3O4. Then Fe3O4@NixSiOymagnetic microspheres with alayered shell were obtained after reaction in an alkali solution containing NiCl2in the autoclave. SEM,HRTEM, XRD, XPS and VSM were used to characterize their morphology, structure, composition andmagnetism. Using as affinity adsorbents to enrich His-tagged proteins, the influence of reaction conditionson the separation effect were investigated, and SDS-PAGE was still used to characterize the separationeffect. Results showed that Fe3O4@NixSiOymagnetic microspheres possessed excellent ability to separateand purify protein, and with the help of magnetic field, the separation speed was fast, and the separationeffect is excellent.(4) Preparation of NiO microspheres for His-tagged protein separationNiO microspheres, with a surface stacked by vertical nanometer piece, were synthesized via asolvothermal method. The influences factors on the morphology and property were investigated. Using asaffinity adsorbent, the influence of different conditions on the separation effect were investigated. Theseparated protein was evaluated by SDS-PAGE and BCA quantitative testing was to verify the results.