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

Published: April 30, 2023

Abstract

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)

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.

Declarações

The authors have nothing to disclose.

Materials

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

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Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate. J. Vis. Exp. (Pending Publication), e21093, doi: (2023).

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