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Logan's E.coli RNAP Purification

This protocol was adapted from Nudler et al. Methods Enzymology. 2003. https://www.sciencedirect.com/science/article/pii/S0076687903710118?via%3Dihub

Contents

Section titled “Contents”
  1. Procedure

1. Procedure

Section titled “1. Procedure”
  1. Streak an LB plate of RL 721 strain (Bio from Landick lab)
  2. Grow O/N cultures of EcRNAP-bio containing cells in LB only.
  3. Innoculate 2L flasks with EcRNAP, grow O/N again in LB only.
  4. Spin down cells and snap freeze at -80C.
  5. Thaw cells in 100mL grinding buffer (300 mM NaCl, 50 mM Tris–HCl, pH 8, 5% glycerol, 10 mM EDTA, 0.1% PMSF, 10 mM mercaptoethanol, 133 µg/mL lysozyme) per 4L pellet in a beaker with a stirbar. Stir for 20mins once ice (I did it in the cold room).
  6. Add Sodium Deoxycholate to 0.2% from a 10% stock in water.
  7. Sonicate 20mL at a time lysate at maximum output for 30s followed by 30s rest. I used the Sonic Dismembrator 60 (Fisher Scientific, Chicago, IL) in the Paull lab. Do this for 5-7 rounds or until not viscous anymore.
  8. Spin the lysate down in a Sorvall JA-20 rotor at 12,000rpm for 30 mins.
  9. Make a 10% branched chain PEI (Polymin P) solution by dissolving it in water.
  10. Add PEI to the supernatant while stirring in a beaker until the final concentration of 0.35%. Continue stirring for 10mins.
  11. Centrifuge in a Sorvall JA-20 rotor at 6000rpm for 5 mins.
  12. Resuspend the pellet in 100mL 0-salt A buffer (25mM Tris pH8, 10% glycerol) + 300mM NaCl.
  13. Centrifuge again in a Sorvall JA-20 rotor at 6000rpm for 5 mins.
  14. Resuspend the pellet in 100mL 0-salt A buffer (25mM Tris pH8, 10% glycerol) + 500mM NaCl.
  15. Centrifuge again in a Sorvall JA-20 rotor at 6000rpm for 5 mins.
  16. Resuspend the pellet in 100mL B buffer (25mM Tris pH8, 10% glycerol, 1M NaCl) and spin in cold room for 1 hour to extract the RNAP.
  17. Centrifuge again in a Sorvall JA-20 rotor at 6000rpm for 5 mins.
  18. Stir the supernatant in a beaker in the cold room. Add ammonium sulfate slowly to 65% w/v (39.8g/100mL). Stir for at least 30 mins.
  19. Centrifuge again in a Sorvall JA-20 rotor at 14,000rpm for 45 mins.
  20. Dissolve the protein in 50mL 0-salt A buffer (25mM Tris pH8, 10% glycerol)
  21. Load the protein onto a 5-mL Hi-Trap Heparin column at 0.5mL/min. Wash with A Buffer (25mM Tris pH8, 10% glycerol,100mM NaCL) until the UV stabilizes. Elute from the column with >60% B.
  22. Collect the fractions and dilute to lower the salt.
  23. Load RNAP onto a Resource Q column. Wash the column and then use a very slow gradient to try to separate out the holoenzyme from the core complex. Collect fractions with most sigma70 subunit and concentrate. Mix with an equal volume of glycerol and store at -20C.
  24. This method provides roughly 2mg of protein per 4L pellet.