IHF Overexpression and Purification

Filutowica, Frimek, and Appelt (1994): Gene 147:149-150

 

  1. Grow starter culture in 500 mL LB-Amp[ (50 mg/mL) at 30 C until OD600 = 0.5, usually 3-3.5 hours.
  2. Induce overexpression by shifting temperature to 42 C. Induce for 3 hours. Every hour, remove 50 mL, combine with protein loading buffer and denature for 5 min. in 90 C water bath. Store samples to run out at -20 C.
  3. After 3 hours at 42 C, pellet culture at 3700 rpm for 30 min. Freeze back pellet at -80 C.
  4. Resuspend pellet in buffer A (0.2 mL/g pellet) with 23 mg/mL Dnase I, 5 mM MgCl2, and 1 mM PMSF.
  5. Sonicate in batches on ice 6 times at 90 W for 30 s. each. Stir between bursts.  Incubate at 4 C for 30 min.
  6. Centrifuge 20 min. at 30,000 g (16,000 rpm).
  7. Add ammonium sulfate to supernatant (0.334 g/mL supernatant) with constant stirring over 10 min.
  8. Stir for 20 min., and then centrifuge 20 min. at 30,000 g (16,000 rpm).
  9. Recover supernatant. Add ammonium sulfate (0.564 g/mL supernatant) with constant stirring. Stir 20 min.
  10. Centrifuge 20 min. at 30,000 g (16,000 rpm).
  11. Dissolve precipitate in buffer A (30 mL/500 mL original culture). Dialyze overnight against 4 L buffer A (dialysis bag with 3500 MWCO)
  12. Filter diazylate through 0.45 mm filter. Take aliquot to determine protein content.
  13. Run through heparin-sepharose column equilibrated with Buffer A. Wash column with Buffer A for 2.25 hr at 25.6 mL/hr.
  14. Elute protein with NaCl gradient in buffer A: 327 mL gradient from 0.1-1.7 M NaCl.
  15. Collect 6.4 mL fractions. Monitor A280. Protein should elute between 0.72-1.06 M NaCl (fractions 24-34).
  16. Add glycerol to 50% and store at -20 C.