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

Overview

In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Protocol

1. Protein Purification by Nickel Affinity Chromatography

1. Purify protein using 1 mL of Nickel-Nitriloacetic Acid (Ni-NTA) resin on a gravity column.
   1. The day before use, equilibrate the column overnight at 4 °C with 2 mL of equilibration buffer (10 mM Tris-HCl pH 7.79, 300 mM NaCl, 50 mM NaH₂PO₄, 0.5 mg/mL lysozyme, 5 mM MgCl₂, 10 mM imidazole, 0.25 mM DTT, 5 mM phenylmethane sulfonyl fluoride (PMSF), 10% glycerol).
   2. The following day bring the column from 4 °C to RT prior to loading the clarified lysate and let it stand for ~2–3 h.
      NOTE: Bringing the column to thermal equilibrium is crucial to avoid air bubbles forming within the column.
   3. Resuspend the pellet in the lysis buffer (10 mM Tris-HCl pH 7.8, 300 mM NaCl, 5 mM MgCl₂, 50 mM NaH₂PO₄, 10% glycerol, 0.5 mg/mL lysozyme, 10 mM imidazole, 0.25 mM DTT and 5 mM PMSF).
   4. Sonicate cells on ice for 8 x 10 s intervals, pausing 30 s between pulses.
   5. Clarify the lysate by centrifugation at 3,080 x g for 30 min at 4 °C using a microcentrifuge.
   6. Prepare lysate with equal volumes of lysis buffer then apply the prepped clarified lysate to the column and collect the flow-through.
   7. Reapply clarified lysate flow-through to the column and collect the secondary flow-through.
   8. Wash the column with wash buffer 1 (10 mM Tris-HCl (pH 7.79), 300 mM NaCl, 5 mM MgCl₂, 50 mM NaH₂PO₄, 30 mM imidazole, 10% glycerol). Collect the flow-through.
      NOTE: Inclusion of 5 mM MgCl₂ in the wash and elution buffers is important for enzymatic activity of the purified protein.
   9. Wash the column with wash buffer 2 (10 mM Tris-HCl (pH 7.79), 300 mM NaCl, 5 mM MgCl₂, 50 mM NaH₂PO₄ and 50 mM imidazole).
   10. Collect the flow-through.
   11. Apply 2 mL elution buffer (10 mM Tris-HCl (pH 7.79), 300 mM NaCl, 50 mM NaH₂PO₄, 10% glycerol and 75 mM imidazole). Collect flow-through in two fractions of 1 mL each.
       NOTE: The column after this step can be stored in equilibration buffer at 4 °C if the same protein will be purified in the next purification assay. The column can be used up to 3 times if stored properly.

Materials

Name	Company	Catalog Number	Comments
Ni-NTA resin	G Biosciences	786-940/941
Pierce Disposable Gravity columns, 10 mL	Thermo Scientific	29924
1 mL Spectra/ Por float-A-lyzer G2 dialysis device (MWCO: 20-kD)	Spectrum	G235033
Ultrasonic processor	Sonics	VC-750
Microcentrifuge with D3024/D3024R rotor	Scilogex