Grants and Contracts Details
Description
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 100nm in diameter, can be synthesized in aqueous solution from a range ofprecllrsors, including nickel, cobalt, and iron (Moumen et a1., 1995; Popplewell et a1., 1995; Zins et at, 1999). These particles are formed by a "soft chemical" approach, primarily precipitation of metal salts in alkaline medium (Zins et a1., 1999) or hydrazine reduction (Wu et a1., 2003) and are stable in aqueous solutions. Cordente and co-workers (Cordente et a1., 2003) found nickel nanoparticles (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. vVhen 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 a1., 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.
Keywords: nanoparticle, transgenic tobacco, protein recovery, engineered proteins
Status | Finished |
---|---|
Effective start/end date | 6/1/04 → 11/30/05 |
Funding
- KY Science and Technology Co Inc: $15,000.00
Fingerprint
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.