ABSTRACT
Magnetic Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads (Ni-ferrosphere beads - NFB) of a core-shell structure were synthesized starting from coal fly ash ferrospheres having diameters in the range of 0.063-0.050 mm. The strategy of NFB fabrication is an oriented chemical modification of the outer surface preserving the magnetic core of parent beads with the formation of micro-mesoporous coverings. Two routes of ferrosphere modification were realized, such as (i) hydrothermal treatment in an alkaline medium resulting in a NaP zeolite layer and (ii) synthesis of micro-mesoporous silica on the glass surface using conventional methods. Immobilization of Ni(2+) ions in the siliceous porous shell of the magnetic beads was carried out via (i) the ion exchange of Na(+) for Ni(2+) in the zeolite layer or (ii) deposition of NiO clusters in the zeolite and silica pores. The final NFB were tested for affinity in magnetic separation of the histidine-tagged green fluorescent protein (GFP) directly from a cell lysate. Results pointed to the high affinity of the magnetic beads towards the protein in the presence of 10 mM EDTA. The sorption capacity of the ferrosphere-based Ni-beads with respect to GFP was in the range 1.5-5.7 mg cm(-3).
Subject(s)
Green Fluorescent Proteins/isolation & purification , Histidine/chemistry , Iron Compounds/chemistry , Nickel/chemistry , Organometallic Compounds/chemistry , Silicon Dioxide/chemistry , Zeolites/chemistry , Animals , Escherichia coli/chemistry , Escherichia coli/cytology , Green Fluorescent Proteins/chemistry , Magnetic Phenomena , Organometallic Compounds/chemical synthesis , Particle Size , Porosity , Scyphozoa , Surface PropertiesABSTRACT
The results of the development and utilization of an affine magnetic sorbent with Ni2+ ions immobilized on coal ash microspheres are reported. The applicability of the material in the isolation of Histag proteins is demonstrated by examples of the recombinant green fluorescent protein from Clytia gregarium and the Ca2+ regulated photoprotein obelin from Obelia longissima. The specific sorption capacity of the sorbent was 2-7 mg/cm3 for medium-size proteins (20-30 kDa). The particles are suitable for chromatography with the presence of chaotropic agents and EDTA. They are easy to manipulate as isolation of a target protein takes 30-35 min. On one hand, the elevated affinity of the sorbent to proteins rich in native histidines may result in a high degree of irreversible sorption; on the other hand, it allows isolation of such proteins without the introduction of artificial polyhistidine tracts.
