Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

To purify recombinant protein fused to poly-histidine tag – a string of histidine residues at one terminus – from a bacterial cell lysate, first, dilute the cell lysate to prevent column clogging during chromatographic run.

Assemble an affinity column containing divalent nickel cations immobilized onto an agarose bead matrix using nitrilotriacetic acid, a chelating agent. Nitrilotriacetic acid binds nickel cations via four coordinate covalent sites, while two sites on the metal ion remain available for interactions with poly-histidine tags in the protein of interest.

Equilibrate the column with suitable buffer to optimize conditions for effective metal-protein interactions. Load the cell lysate onto the column.

Recombinant proteins, carrying accessible terminal histidine residues, form coordination bonds with nickel cations due to the metal ion's high affinity for imidazole, the side-chain of histidine, and bind to the matrix. The high number of consecutive histidines in the recombinant protein strengthens its attachment to the matrix.

In comparison, proteins lacking histidine residues do not adhere to the column and elute in flow-through. Wash the column with low-concentrated imidazole buffer.

Imidazole competes with weakly bound protein contaminants that may have nonspecifically adhered to the matrix via histidine residues in their polypeptide chain, displacing them and facilitating elution. Wash the column with highly concentrated imidazole buffer to dissociate the strongly-bound histidine-tagged recombinant proteins from nickel ion chelate, eluting them in flow-through. 

Collect the purified recombinant protein fraction for further analysis.