Purification of Strep-tagged proteins using PureCube HiCap Streptactin MagBeads

 

This protocol describes the generation of a cleared lysate from an E. coli cell pellet and the subsequent purification of Strep-tagged proteins under native conditions using PureCube HiCap StrepTactin MagBeads. Reagent amounts given apply to 10 mL IPTG-induced bacterial culture of a well-expressed protein (approximately 10–50 mg/L). If other culture volumes are processed or protein expression is higher or lower, reagent volumes may need to be adjusted. Magnetic bead purification is easily scalable. To minimize unspecific binding and reduce cost, the volume magnetic bead suspension used should be adjusted to the expression level of interest. See Table 1 for more details. 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 Streptagged target protein is purified from cleared lysate under native conditions in a bind-wash-elute procedure.

Buffer compositions - Protein purification protocol

Lysis Buffer, 10 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
TRIS base, pH 8.0 100 mM 121.14 1 M 60.57 g/ 500 mL 1 mL
NaCl 150 mM 58.44 5 M 146.1 g/500 mL 300 µL
Protease inhibitor 1x - follow supplier‘s instructions
Lysozyme 1 mg/ml - - - 10 mg
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0. Add protease inhibitor and lysozyme directly before use.

Wash Buffer, 50 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
TRIS base, pH 8.0 100 mM 121.14 1 M 60.57 g/ 500 mL 5 mL
NaCl 150 mM 58.44 5 M 146.1 g/500 mL 1.5 mL
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0.

Elution Buffer, 10 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
TRIS base, pH 8.0 100 mM 121.14 1 M 60.57 g/ 500 mL 1 mL
NaCl 150 mM 58.44 5 M 146.1 g/500 mL 300 µL
Desthiobiotin 2.5 mM 214.26 25 mM 53 mg/10 mL 1 mL
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0. Add desthiobiotin directly before use.

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.
   
A.Protein purification protocol für native conditions:
 
 
  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.
  3. Add 6 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
  4. Incubate for 30 min on ice.
  5. Centrifuge the lysate for 30 min at 10,000xg and 4˚C. Collect the supernatant. Note: The supernatant contains the soluble proteins and is the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
  6. Resuspend the PureCube HiCap StrepTactin MagBeads by vortexing. Transfer 200 μL of the 5% magnetic bead suspension into a conical microcentrifuge tube (or the volume adjusted to the expression level
  7. Add 1 mL Wash Buffer and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
  8. Repeat step 7 twice.
  9. Pipet 1 mL of the cleared lysate onto the equilibrated magnetic beads, and incubate the lysate-magnetic bead mixture at 4˚C for 1 h on an end-over-end shaker
  10. Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant. Note: This is the flow-through fraction
  11. 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. Note: These are the wash fractions.
  12. Repeat step 11 twice
  13. Elute the Strep-tagged protein using 100 μL Elution Buffer.
  14. Repeat step 13 five times. Collect each elution fraction in a separate tube and determine the protein concentration of each fraction. Note: These are the elution fractions.Optional: Incubate the resin for 15 min in Elution Buffer before collecting the eluate to increase protein yields
  15. Optional: Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate samples at 46˚C for 30 min in preparation for SDS-PAGE analysis.
StrepTactin® and Strep-tag® are trademarks of IBA GmbH. PureCube StrepTactin protein is manufactured by IBA GmbH under German Patent Application No. 42 37 113.9 entitled "Fusion peptides with binding activity for streptavidin"
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