Utilization of Submicron and Nano-sized Nickel Particles to Facilitate Recovery of Histidine-tagged Proteins

摘要

Recently there have been significant advances in the development of transgenic tobacco (or other plants), 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. One of the final downstream purification options is torecover the histidine-tagged target protein with a Ni~(2+) metal affinity chromatography column, where chromatography steps typically require high pressure and expensive matrix regeneration. In this study, submicron and nano-sized nickel particles are utilized to perform the separation obtained with Ni~(2+)metal affinity chromatography columns. Histidine-tagged green fluorescent protein (GFP) was the target protein used because of the direct relationship between the fluorescence reading and the amountof protein present. Two separate set-ups were tested to magnetically collect the nickel/protein complex. In the first system, unwanted solution was removed via gravity while the nickel/protein complex remained in the tubing magnetically, subsequently thenickel/protein complex was removed with a washing after removing the magnets. In the second system, the solution was placed in a cuvette, a magnet was placed at the bottom of the cuvette and the unwanted solution was removed via a pipetter from the top.The first system recoveries were remarkably low (1-3.5%), while the second system recoveries were as high as 85%. Utilizing the second system, nine different experimental conditions were studied to be compared to a base case. Conditions varied by amountand type of nickel powder, ratio ofretentate volume to total cuvette volume, and pH. The highest enrichment (7.5) was found with the base case conditions, while the highest recovery (85%) was found at a pH of 6.5 (as opposed to the 7.5 associated with the base case).