SsDddA Purification with FPCL 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

Equipment

  • Shaker incubator
  • Centrifuge capable of up to 40,000 × g
  • Sonicator with a 13 mm probe
  • FPLC system
  • Spectrophotometer

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.