Sauer:GST-sFtsH purification

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Contents

GST-sFtsH purification

Expression

  1. Inoculate overnight from glycerol stock.
  2. Wash overnight growth (spin down, resuspend, repeat) before inoculating 2x1L TB/Amp
  3. Grow to OD 1, induce with 0.5mM IPTG.
  4. Let grow ~2h. Store at -80.

Buffers

Lysis Buffer:

  • 50mM TrisHCl pH 8
  • 1mM EDTA
  • 1mM DTT

Glutathione Ag Elution Buffer:

  • 50mM Tris HCl
  • 10mM reduced glutathione
  • 400mM KCl

Generic buffer (GB) for this purification

  • 50mM Tris-HCl pH 8
  • 150mM KCl
  • 10mm MgCl2
  • 10uM Zinc acetate
  • 1Mm EDTA
  • 1Mm 2mM DTT

Purification

  1. Lyse cells by chemical lysis in the presence of protease inhibitor cocktail.
  2. Centrifuge.
  3. Resuspend in lysis buffer
  4. Ammonium Sulfate cut- add sat AmSO4 to 60%. (make sure to add slowly, with stirring, in the cold room) Let incubate 1h +
  5. Centrifuge, 20m by 12krpm
  6. Resuspend in lysis buffer
  7. PD10 to remove excess salt
  8. Add Glutathione Agarose resin. Let incubate 30m.
  9. Wash column with 10 volumes lysis buffer
  10. Elute 2x with Glutathion Ag Elution Buffer
  11. Buffer Exchange eluted protein to remove excess glutathione
  12. Precision Protease (40uL per L starting culture) 40h
  13. Incubate with Glutathione Agarose.
    1. Collect flow through + 1-2 CV of wash.
  14. Concentrate
  15. Superdex 300 column

Concentrate/buffer exchange in GB

Elute Glutathione Agarose column with EB- keep to ensure success in cleavage

S300 size-exclusion column (S200 was used here in the published protocol, but the protein if hexameric should be in the exclusion volume with the S200)

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