Knight:Purification of His-tagged proteins/Denaturing

Overview

Denaturing purifications can often lead to better purity and yield.

Materials

Lysis and column equilibration buffer

• 8 M urea
• 100 mM NaH₂PO₄
• 10 mM Tris Cl
• 10 mM imidazole (recommended by Kathleen, 9/27/2006)
• pH 8.0
• 1 mM TCEP (add just before use)

Wash buffer (Qiagen buffer C)

• 8 M urea
• 100 mM NaH₂PO₄
• 10 mM Tris Cl
• 10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?)
• pH 6.3
• 1 mM TCEP (add just before use)

Elution buffer (Qiagen buffer E)

• 8 M urea
• 100 mM NaH₂PO₄
• 10 mM Tris Cl
• 10 mM imidazole (recommended by Kathleen, 9/27/2006, also use here?)
• pH 4.5
• 1 mM TCEP (add just before use)

Notes

• Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H ₂O you start with (maybe 50% of final volume)
• Kathleen suggested supplementing the lysis buffer with 10mM imidazole to prevent nonspecific protein binding to the column. Perhaps supplement the other buffers as well?
• The urea should be freshly prepared and deionized prior to use.
• The buffers should each be degassed before use.
• TCEP is not particularly stable in phosphate buffers, especially at neutral pH. Therefore, if TCEP is to be used in phosphate buffers, prepare the working solution immediately before use.

Procedure

Grow and pellet cultures

1. Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
2. The following morning, dilute back the culture 1:50 to the appropriate culture volume (which depends on the expected yield of the protein).
   • Since I don't know what yield to expect, I arbitrarily did 50mL cultures assuming that my protein yield would be low and let it grow most of the day. I try to catch the cultures around OD600nm 0.6.
3. Harvest the cells by centrifugation at 4000 x g for 15 mins.
   • The Qiagen protocol didn't specify a temperature so I did 4°C.
4. Decant supernatant.
5. The cell pellet can be stored at -70°C or processed immediately.
   • I stored the pellet at -80°C. Long term storage at -80°C might affect protein recovery???

Prepare solutions

1. Prepare 10M Urea.
2. Deionize the urea solution.
3. Prepare lysis, wash and elution buffers (everything except reducing agent).
4. Verify pH of lysis, wash and elution buffers. Adjust if necessary.
   • Dissociation of urea can lead to changes in pH. The pH definitely needs to be checked prior to using the solutions.
5. Degas lysis, wash and elution buffers by placing under vacuum for one hour.
   • Tom recommends doing the degassing step last.
6. Add TCEP to 1mM final concentration.
   • TCEP is not stable in phosphate buffers at neutral pH.
7. Verify that pH hasn't changed.
   • It is not known whether TCEP affects pH. Degassing can affect pH in some cases.

Purify protein

1. Thaw pellet for 30 mins.
   • Can thaw at room temperature since the next step happens at room temperature. However, proteases will be less active if the pellet is thawed on ice.
   • The Qiagen protocol calls for 15 mins, but it was still frozen after 15 mins so I let it thaw for 30 mins.
2. Transferred to 2mL eppendorf tube.
3. Resuspend in 1mL Lysis Buffer (see above).
4. Add half or 1/4 protease inhibitor pellet.
5. Incubate cells with agitation for 1 hr at room temperature.
   • Use an orbis shaker on the bench to do this temp (usually kept in 37° incubator). Note that the shaker moves during shaking.
6. Centrifuge lysate at 10000 x g for 30 mins at room temperature.
7. Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
8. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid to remove equilibration buffer.
9. Save 20 μL cleared lysate.
10. Load 600 μL cleared lysate to Ni-NTA column.
11. Centrifuge Ni-NTA column 5 mins at 700 x g with closed lid.
    • I typically repeat this step to load the rest of my cleared lysate.
    • Save flow through.
12. Add 600 μL wash buffer to Ni-NTA column.
13. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Save flow through.
14. Add 600 μL wash buffer to Ni-NTA column.
15. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Save flow through.
16. Tranfer to clean 1.5mL eppendorf tube.
17. Add 200μL elution buffer.
18. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Most of the protein should elute in this elution step.

Notes

• Sauer lab uses a Qiagen Ni-NTA resin but this protocol uses spin columns. (Smaller scale purification).
• Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields.
• A lower pH may be needed for elution. For instance, aggregates should elute at pH 4.5 whereas monomers generally elute at pH 5.9.
• Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column.
• 20 year old spin columns don't work.  :)
• These columns can tolerate up to 20mM β-mercaptoethanol or 1mM dithiothreitol. Some people also seem to use 0.5-1mM TCEP with these columns. TCEP appears to more compatible with NTA columns for his-tagged protein purification.
• Even when doing denaturing purifications, add 10mM imidazole to solutions to help with washing out non His tagged proteins.

Troubleshooting and optimization

• Deionize urea solutions prior to use.
• Degas all buffers.

References

1. Sauer:Purification of His-tagged proteins/Denaturing prep

2. Qiagen Ni NTA Spin Kit manual