DddI Purification Protocol

This protocol for the expression and purification of DddI was compiled and optimized by Devaraja G. Mudeppa (Stergachis Lab, Division of Medical Genetics, University of Washington).

Buffers & Reagents

Buffer A

ComponentFinal Concentration
Tris-HCl, pH 7.850 mM
NaCl600 mM
Glycerol10%
Imidazole10 mM
Triton X-1000.1%
2-Mercaptoethanol5 mM

Wash Buffer

ComponentFinal Concentration
Tris-HCl, pH 7.850 mM
NaCl500 mM
Imidazole30 mM
2-Mercaptoethanol5 mM

Buffer B

ComponentFinal Concentration
Tris-HCl, pH 8.050 mM
NaCl600 mM
Imidazole500 mM
Glycerol10%

Storage Buffer

ComponentFinal Concentration
Tris-HCl, pH 7.850 mM
NaCl500 mM
DTT1 mM
Glycerol10%

Additional Reagents

  • IPTG stock: 1 M in water
  • Kanamycin stock: 100 mg/mL in water

DddI Expression

  1. Transform BL21(DE3) with pColDuet_nHis_SsDddI, plate on LB-kan agar (50 µg/mL). Incubate overnight at 37 °C.
  2. Inoculate a single colony from the plate into 12 mL LB containing 6 µL of kanamycin stock (100 mg/mL).
  3. Grow overnight at 37 °C with shaking at 250 rpm.
  4. Add 10 mL of the overnight culture to 1 L of LB containing 500 µL of kanamycin stock (100 mg/mL).
  5. Grow at 37 °C with shaking at 250 rpm for approximately 5 hours.
  6. Measure OD600 and adjust to OD600 ≈ 0.6 if necessary, using fresh LB medium.
  7. Briefly cool the culture by placing it on ice for a few minutes.
  8. Add 300 µL of 1 M IPTG to 1 L of culture to achieve a final concentration of 0.3 mM.
  9. Incubate at 25 °C with shaking at 250 rpm for approximately 18 hours.
  10. Pellet cells (4000 × g, 10 min) and store the cell pellet at −80 °C.

DddI Purification

Note: All purification steps were performed at 4 °C. Unless noted, the flow rate was 2 mL/min.

  1. Thaw the 1 L culture pellet at room temperature for 10 minutes (skip this step if using a fresh pellet).
  2. Add 35 mL of Buffer A containing two cOmplete Mini EDTA-free protease inhibitor tablets (Millipore-Sigma; Cat# 11836170001). Vortex vigorously for 2–3 minutes.
  3. Transfer the resuspended cells to a 50 mL Falcon tube and vortex again to eliminate lumps.
  4. Sonicate with a 13 mm probe for a total time of 10 min (30 s on/30 s off cycles) at 30% amplitude.
  5. Centrifuge the lysate at 30,000 × g for 30 min.
  6. Load the supernatant onto a 5 mL HisTrap Excel column (Cytiva; Cat# 17371206), which has been pre-equilibrated with 5 column volumes (CV) of Buffer A.
  7. Wash the column with 5 CV of Buffer A, then 10 CV Wash Buffer.
  8. Elute column-bound DddI by applying a 5 CV (25 mL) gradient from 100% Wash Buffer to 100% Buffer B.
  9. The protein eluted between ~30% and ~70% was collected.
  10. Concentrate eluted DddI using a 15 mL 3 kDa MWCO Amicon filter (Millipore Sigma; Cat# UFC900308).
  11. Perform buffer exchange by diluting the protein to 15 mL with Storage Buffer and reconcentrating to 1 mL; repeat this step 3 times.
  12. Quantify purified DddI using the Bradford assay. Adjust DddI concentration to 1000 µM using Storage Buffer. Verify purity using 4–20% SDS-PAGE.
  13. Store the purified DddI in required aliquots at −80 °C. The DddI is stable for at least 5 freeze-thaw cycles.