SsDddA Purification Protocol

This protocol for the expression and purification of SsDddA 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%
Triton X-1000.1%

Denaturation Buffer

ComponentFinal Concentration
Tris-HCl, pH 7.850 mM
Imidazole20 mM
NaCl500 mM
Guanidine HCl (ThermoFisher, Cat# 24110)6 M
2-Mercaptoethanol5 mM

Renaturation Buffer

ComponentFinal Concentration
Tris-HCl, pH 7.850 mM
NaCl500 mM
ZnCl₂10 µM
2-Mercaptoethanol10 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
ZnCl₂10 µM
DTT1 mM
Glycerol10%

Additional Reagents

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

DddA–DddI Complex Expression

  1. Transform BL21(DE3) with pColDuet-DddA-DddI, 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 RCF, 10 min) and store the cell pellet at −80 °C.

DddA 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).
  3. Vortex vigorously for 2–3 minutes.
  4. Transfer the resuspended cells to a 50 mL Falcon tube and vortex again to eliminate lumps.
  5. Sonicate with a 13 mm probe for 5 minutes (30 s on/30 s off cycles) at 30% amplitude; total sonication time is 10 min.
  6. Centrifuge the lysate at 40,000 × g for 1 hour.
  7. 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.
  8. Wash the column with 5 CV of Buffer A, then 5 CV Renaturation Buffer.
  9. Denature column-bound DddA/DddI by applying a 10 CV gradient from 100% Renaturation Buffer to 100% Denaturation Buffer.
  10. Maintain the column in Denaturation Buffer for an additional 5 CV.
  11. Renature DddA by applying a 15 CV gradient from 100% Denaturation Buffer to 100% Renaturation Buffer.
  12. Maintain the column in Renaturation Buffer for an additional 10 CV at 1 mL/min.
  13. Elute DddA using 5 CV of Buffer B.
  14. Pool protein-containing fractions (based on A280) and concentrate to 1 mL using a 15 mL 3 kDa MWCO Amicon filter (Millipore Sigma; Cat# UFC900308).
  15. Perform buffer exchange by diluting the protein to 15 mL with Storage Buffer and reconcentrating to 1 mL; repeat this step 3 times.
  16. Quantify purified DddA using the Bradford assay. Adjust DddA concentration to 100 µM using Storage Buffer. Verify purity using 4–20% SDS-PAGE.
  17. Store the purified DddA in required aliquots at −80 °C. The DddA is stable for at least five cycles of freeze-thaw.