DOI: 10.2144/000113301
Abstract
The HaloTag® Protein Purification System is based on a unique protein tag, HaloTag®, which allows covalent, efficient, and specific capture of proteins expressed in Escherichia coli. HaloTag® was engineered from a bacterial dehalogenase to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. The following is a sample of one protocol for the HaloTag Protein Purification System. The system can also be used in an all “batch method”. References to specific detail can be found in the full protocol (download the PDF).
Batch/column combination method for HaloTag® Protein Purification
This protocol is for cells prepared from 50 mL cell culture with 1 mL settled
HaloLink™ Resin (Promega Corp, Madison, WI).
HaloLink™ Resin equilibration
Equilibrate the HaloLink™ Resin as described in Section 4.D.
Binding/immobilization
1. Bind the protein to the resin as described in Section 4.D, steps 8–9.
2. Load all resin onto column, drain by gravity, collect the flowthrough (FT),
and store on ice for further analysis. Keep some solution on top of the
resin to avoid drying the resin.
Wash
3. Add a total of 20 mL HaloTag® Protein Purification Buffer (20× column
volume) and drain by gravity.
4. After all the buffer has drained, close the end of the column tightly to
avoid drying the resin.
TEV protease cleavage/elution
5. Add 1 mL (1 column volume) cleavage solution onto the settled resin.
6. Open the end of the column and let ~1 mL of the solution run out (1 column
volume) Close the end of the column tightly.
7. Cap the top of the column to avoid evaporation and incubate at room
temperature for 1 h.
8. During the incubation, label two 1.5-mL microcentrifuge tubes:
S (supernatant) and S-TEV (supernatant + TEV).
• Add 50 µL cell lysate supernatant saved from Section 4.C and 10 µL HaloTag®
Protein Purification Buffer to one tube (tube S).
• Add 50 µL cell lysate supernatant and 10 µL cleavage solution to the other
tube (tube S-TEV) and mix well. This tube will be used to evaluate cleavage
and find the size of the cleaved protein following SDS-PAGE.
• Incubate the two tubes at room temperature for 1 h and then place on ice.
9. Add 2 mL HaloTag® Protein Purification Buffer to the column, being careful
not to disturb the resin.
10. Open the end of the column and collect a total of ~2 mL (2 column volumes)
elution into a 15-mL conical tube. This will be Elution 1 (E1).
11. Save 100 µL of E1 in a microcentrifuge tube, and store on ice for analysis.
TEV protease removal
12. Thoroughly resuspend the HisLink™ Resin.
13. Add 50 µL of 50% HisLink Resin into the tube from step 10. Incubate the
tube on a rotator at room temperature for 20 min.
14. Spin at 1,000× g for 5 minutes and transfer supernatant to
another tube. This is your purified protein. Remove a small fraction for
analysis. This is elution 2 (E2).
Gel analysis
15. Thoroughly resuspend the HisLink™ Resin.
16. Add 50 µL 50% HisLink Resin into the tube from step 10. Incubate the tube
on a rotator at room temperature for 20 min.
17. Spin at 1,000× g for 5 min and transfer supernatant to another
tube. This is your purified protein. Remove a small fraction for analysis.
This is elution 2 (E2).
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