Knight:Purification of His-tagged proteins/Native

Overview

Native purifications permit purification of the folded protein.

Materials

• Lysozyme
• RNase A (optional)
• DNase A (optional)

Lysis and column equilibration buffer

• 5mM imidazole
  • can vary between 1mM and 20mM

Procedure

1. Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
2. The following morning, dilute back the culture 1:60 to the appropriate culture volume (which depends on the expected yield of the protein).
   • Maybe try 200mL cultures.
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.
6. Thaw for 15 mins on ice.
   • May take longer than 15 mins.
7. Transferred to 2mL eppendorf tube.
8. Resuspend in 2-5mL Lysis Buffer (see above) per gram wet weight.
   • The lysis buffer contains 10mM imidazole to reduce untagged, contaminating proteins. The imidazole concentration can be reduced to either 1-5 mM or increased to 20 mM as appropriate.
9. Add lysozyme to 1 mg/mL concentration.
10. Incubate cells cells on ice for 30 mins.
11. Optional: If the lysate is very viscous, add 10 μg/mL RNase A and 5 μg/mL DNase I and incubate on ice for 10-15 mins.
    • Or draw the lysate through a narrow gauge blunt-ended syring needle several times.

1. Centrifuge lysate at 10000 x g for 30 mins at room temperature.
2. Add 600 μL lysis buffer to Ni-NTA column to equilibrate.
3. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid to remove equilibration buffer.
4. Save 20 μL cleared lysate.
5. Load 600 μL cleared lysate to Ni-NTA column.
6. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
   • Repeat this step to load the rest of my cleared lysate?
   • Save flow through.
7. Add 600 μL wash buffer to Ni-NTA column.
8. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
   • Save flow through.
9. Add 600 μL wash buffer to Ni-NTA column.
10. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Save flow through.
11. Tranfer to clean 1.5mL eppendorf tube.
12. Add 200μL elution buffer.
13. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Most of the protein should elute in this elution step.
14. Tranfer to clean 1.5mL eppendorf tube.
15. Add 200μL elution buffer.
16. Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
    • Just in case.

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.
• 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.  :)

References

1. Qiagen Ni NTA Spin Kit manual