Purification of GST-tagged proteins using PureCube Glutathione MagBeads

 

This protocol describes the generation of a cleared lysate from an E. coli cell pellet and the subsequent purification of GST-tagged proteins under native conditions using PureCube Glutathione MagBeads. Reagent amounts given apply to 10 mL IPTG-induced bacterial culture of a well-expressed protein (approximately 10–50 mg/L). In this protocol, cell lysis is done using lysozyme because it is an inexpensive and efficient method for cells that have been frozen. However, lysis methods using detergents (e.g., CHAPS) can also be used. The GST-tagged target protein is purified from cleared lysate under native conditions in a bind-wash-elute procedure.

Buffer compositions - Native protein purification protocol

Lysis Buffer, 100 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Tris-HCl, pH 7.4 125 mM 121.14 0.5 M 30.29 g/500 mL 25 mL
NaCl 150 mM 58.44 5 M 146.1 g/500 mL 3 mL
DTT 1 mM 154.25 1 M 14.6 g/100 mL 200 µL
EDTA 1 mM 292.24 0.5 M 1.54 g/10 mL 100 µL
Lysozyme 1 mg/mL - 100 mg/mL 1 g /10 mL 1 mL
Triton X-100 1% (v/v) - 100%(v/v) - 1 mL
Protease inhibitor 1x - - - 2 tablets
Instructions: Prepare a 0.5 M Tris-HCl stock by dissolving Tris base in 400 mL deionized water, adding HCl to a pH of 7.4, and adding water to a final volume of 500 mL. Lysis buffer should always be prepared fresh.

Wash Buffer, 100 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Tris-HCl, pH 7.4 125 mM 121.14 0.5 M 30.29 g/500 mL 25 mL
NaCl 150 mM 58.44 5 M 146.1 g/500 mL 3 mL
DTT 1 mM 154.25 1 M 1.54 g/10 mL 100 µL
EDTA 1 mM 292.24 0.5 M 14.6 g/100 mL 200 µL
Instructions: Optimal buffer conditions may vary depending on the protein of interest. Proteins may require addition of protease inhibitor cocktail, EDTA, 1-5 mM DTT, 1% BSA, or detergents such as 0.5-1% Igepal CA-630 (Nonindet P-40) or 0.5-1% Tween-20.

ATP Buffer (optional), 10 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Tris-HCl, pH 7.4 50 mM 121.14 0.5 M 30.29 g/500 mL 1 mL
ATP 2 mM/td> 551.14 100 mM 551 mg/1 mL 200 µL
MgSO4 10 mM 120.37 1 M 1.24 g/ 1 mL 100 µL
Instructions: Add water to 10 mL. Always prepare fresh.

Elution Buffer, 10 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Tris-HCl, pH 7.4 125 mM 121.14 0.5 M 30.29 g/500 mL 2.5 mL
NaCl 150 mM 58.44 5 M 146.1 g/ 500 mL 300 µL
Triton X-100 0.1% (v/v) - 100% (v/v) - 10 µL
Reduced glutathione 50 mM 307.32 - - 154 mg
DTT 1 mM 154.25 1 M 1.54 g/10 mL 10 µL
Instructions: Dissolve in 8 mL water, stir until the reduced glutathione is completely dissolved. Depending on the protein‘s requirements, set the pH to 7.4-8.0 using NaOH, then add water to 10 mL. Always prepare fresh.

5X SDS Page Buffer, 10 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Tris-HCl, pH 6.8–7.0 300 mM 121,14 1 M 121,14 g/ 1 L 3 mL
Glycerol 50% (v/v) - 100% (v/v) - 5 mL
SDS 5% (w/v)) - - - 0.5 g
Bromophenol blue 0.05% (w/v) - 4% - 125 μL
DTT 250 mM 154.25 1 M 1.54 g/ 10 mL 125 μL/aliquot
Instructions: Make sure to prepare a 1 M Tris-HCl stock by dissolving Tris base in 500 mL deionized water, adding HCl to a pH of 6.8–7.0, and adding water to a final volume of 1 L. For the SDS-PAGE Buffer, mix all components listed except DTT and add water to a total of 10 mL. Freeze 20 aliquots (375 µL each) at –20˚C. Before use, add DTT to the needed single aliquots.

Table 1. Magnetic bead suspension volumes suitable for given protein expression levels

 
Protein expression levelAmount of GST-tagged protein per 1 mL cultureAmount GSTtagged protein per 10 mL* cultureVolume 25% magnetic bead suspension per 10 mL cultureMinimum elution volume per 10 mL culture
>0.5 mg/L >0.5 μg >5 μg >2 μL* >25 μL
1 mg/L 1 μg 10 μg 4 μL* 25 μL
5 mg/L 5 μg 50 μg 20 μL 50 μL
10 mg/L 10 μg 100 μg 40 μL 100 μL
50 mg/L 50 μg 500 μg 200 µL 500 μL
*Note: For easier handling, dilute the magnetic bead suspension in PBS buffer pH 7.4 before use.
   
A.Protein purification protocol:
 
 
  1. Thaw the E. coli cell pellet on ice. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
  2. Resuspend the cell pellet in 1 mL Lysis Buffer. Add 30 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
  3. Incubate for 30 min on ice, if necessary for protein stability. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
  4. Centrifuge the lysate for 30 min at 10,000xg and 2-8 °C. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  5. Resuspend the PureCube Glutathione MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic beads suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
  6. Add 500 μL Lysis Buffer to the Glutathione MagBeads and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
  7. Pipet 1 mL of the cleared lysate onto the equilibrated MagBeads, and incubate the mixture at 4 °C for 1 h on an end-over-end shaker.
  8. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant.
  9. Remove the tube from the magnet. Add 500 μL Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
  10. Repeat step 10 twice. 12: Optional: To remove contaminants such as chaperones, perform an additional wash step with ATP buffer.
  11. Elute the GST-tagged protein using 100 μL Elution buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
  12. Repeat step 12xxx Collect each elution fraction in a separate tube.
  13. Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate the sample at 46 °C for 30 min in preparation for SDS-PAGE analysis.
  14. Optional: Perform Western Blot experiment using GST Antibody.
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