Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography
Transcrição
Take a suspension of engineered bacteria expressing recombinant self-assembling protein nanoparticles with polyhistidine tags.
Sonicate to release intracellular components.
Centrifuge to collect the supernatant containing protein nanoparticles. Dilute it with an imidazole-free buffer.
Take an affinity chromatography column containing agarose beads with immobilized nickel cations.
Add a buffer with a low imidazole concentration for column equilibration.
Add the bacterial lysate onto the column.
During the run, the polyhistidine on protein nanoparticles binds strongly with nickel cations.
Meanwhile, contaminants like bacterial lipopolysaccharides and other proteins bind weakly to the column.
Wash with a buffer containing a low imidazole concentration to remove the weakly bound proteins.
Introduce isopropanol to remove lipopolysaccharides and then wash.
Next, introduce a buffer with an intermediate imidazole concentration. The imidazole competes with histidine for binding to the nickel cation, releasing bound protein nanoparticles.
Next, add a buffer containing a high imidazole concentration to completely elute the protein nanoparticles.
Collect the purified protein nanoparticles for further application.
