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Utilization of Nickel Nanoparticles to Facilitate the Recovery of Histidine Tagged Proteins
University of Kentucky Recently, there have been significant advances in the development of transgenic tobacco, which can be used as a solar powered bioreactor, producing commercially attractive pharmaceuticals or enzymes. Several plant biotech companies have identified valuable product targets, but there is a need for acceptable production practices and industrial scale process development before transgenic plants can provide a new market for growers. The cost of protein recovery and purification may be the determining factor in whether a product is economically viable. One of the final downstream purification options is to recover a histidine-tagged target protein with a Ni2+ metal affinity chromatography column, where chromatography steps typically require high pressure and expensive matrix regeneration. We propose to use nickel nanoparticles to bind histidine-tagged proteins, facilitating their removal by magnetic collection. Metallic nanoparticles, ranging from a few nanometers to 100 nm in diameter, can be synthesized in aqueous solution from a range of precursors, including nickel, cobalt, and iron (Moumen et al., 1995; Popplewell et al., 1995; Zins et al., 1999). These particles are formed by a "soft chemical" approach, primarily precipitation of metal salts in alkaline medium (Zins et al., 1999) or hydrazine reduction (Wu et al., 2003) and are stable in aqueous solutions. Cordente and co-workers (Cordente et al., 2003) found nickel nanopartic1es (d~4.5 nm or larger) retain the same magnetization value as bulk nickel, allowing for collection by magnetic probe. It should be possible to utilize these nickel nanoparticles to achieve the same separation obtained with Ni2+ metal affinity chromatography columns, but with less cost and without protein denaturation. In this study, histidine-tagged proteins suspended in a tobacco extract solution will be complexed with Ni2+ ions on the surface of a nickel nanoparticle. When a magnetic collector is applied to the solution, only the nickel nanoparticles and the histidine tagged protein will be recovered. The histidine-tagged proteins will then be removed from the nanoparticle's surface by elution with imidiazole followed by dialysis (Lige et al., 1998), resulting in the removal of the unwanted tobacco proteins and a reduction in the volume of the product solution. The parameters that will be investigated include solution components, concentrations, and nickel particle size and conditioning.
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