Sauer: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.
 * 14) 	Collect flow through + 1-2 CV of wash.
 * 15) Concentrate
 * 16) 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)