Sauer:Purification of His-tagged proteins/Denaturing prep

Introduction

This is a general protocol for purification of His₆-tagged proteins under denaturing conditions. Note that you may need to modify this protocol to fit your specific needs. This protocol is a modified version of that provided with Ni²⁺-NTA resin from Qiagen.

In general, you will get a protein of higher purity if you purify it under denaturing conditions. Fewer cellular proteins stick non-specifically to the resin under denaturing conditions. However, purifying under denaturing conditions requires that you have a protocol for refolding your protein that results in a native structure. The protocol below involves refolding the protein while it is still bound to the resin. It is thought that this procedure works better than refolding in solution because the bound proteins are physically separated from one another on the resin and, thus, should have a lower tendency to aggregate.

Lysis

The amount of lysis buffer you use will depend on the number of cells you have. The following works well for cells harvested from a 1 L culture, and I never pay attention to the density of the culture.

1. Resuspend cell pellet in 10 mL lysis buffer.
2. Freeze at -80 ˚C for at least 30 min.
3. Thaw and bring lysis buffer volume to 40 mL.
4. Allow to lyse at room temperature for 30 min.
5. Spin to pellet the cellular debris at 14000 rpm for 30 min in a SA600 or similar rotor.
6. Retain the supernatent.

Purification

The amount of Qiagen Ni²⁺-NTA resin (or similar product) you use depends on the amount of protein you have in your culture. Less is typically better. The protocol below works for most proteins that were grown in a 1 L culture.

1. Add 1 mL of 50% Ni²⁺-NTA resin slurry to lysate.
2. Bind at room temperature, shaking gently, for 30 min–1 hr.
3. Spin at low speed to pellet the resin.
4. Remove supernatent and save (this is your flowthrough).
5. Add 7 mL of lysis buffer to the resin and transfer the resin to a small column. (I like these columns from Biorad.)
6. Collect wash.
7. Wash two times with 7 mL of wash buffer 1 (collect).
8. Wash with a total of 50 mL of wash buffer 2 (collect).
9. Elute four times with 1 mL of elution buffer (collect each separately).
10. Run a small amount of each elution (10 µL) and some of the washes, if desired, on an SDS-polyacrylamide gel to assess purity,
11. Combine elutions, concentrate your protein, and buffer exchange as necessary. Note that further columns may be necessary to achieve desired purity.

Buffers

Lysis buffer

100 mM NaH₂PO₄

10 mM Tris base

6 M guanidine hydrochloride

10 mM imidazole

Adjust to pH 8

Wash buffer 1

100 mM NaH₂PO₄

150 mM NaCl

8 M urea

20 mM imidazole

Adjust to pH 8

Wash buffer 2

50 mM NaH₂PO₄

500 mM NaCl

20 mM imidazole

Adjust to pH 8

Elution buffer

50 mM NaH₂PO₄

500 mM NaCl

250 mM imidazole

Adjust to pH 8