Sauer:FtsH purification

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Wild-type FtsH Expression and Purification:

(From Herman et al. 2003)

E.Coli strain JM105 pBHB1

Purification for 4L.

MW ~ 71kD

Expression

  1. Grow in TB/Mg2+ with Chloroamphenicol to OD600 0.6 at 34°C

Mg 2+ - 5mM, therefore 5mL of 1M per flask

  1. Induce with L-arabinose (0.4%) and let grow 2-3hrs. (0.4% = 4g/L)
  2. Spin down, (30min x 4k) and resuspend in buffer A.

Try cranking the salt concentration here

During purification- have CI-ssrA present or some such.

Possible point to freeze cells overnight.

Purification

  1. Lyse. (Try using lysozyme + sonication)
  2. Centrifuge 10min x 4000rpm
  3. Collect supernatant
  4. Spin down supernatant 40 000 rpm

40k rpm requires the ultracentrifuge, Baker lab goal here is about 150 000 x g

  1. Solubilize pellet in buffer B

Try letting sit in Buffer for ~1hr in cold room. May not have fully solubilized.

  1. Centrifuge at 40 000 rpm
  2. Load supernatant onto FFQ column
  3. Elute with linear gradient from 10mM to 1M NaCl in buffer C

FFQ- Fast Flow Sepharose Q, approximately

  1. Load fractions with FtsH (as determined by gel?) onto Superose 12
  2. Elute with buffer C

Superose 12 ~ equivalent to Sephacryl 300

  1. Load fractions with FtsH onto Mono P column
  2. Elute with linear gradient from 10mM to 1M NaCl in buffer C

Mono P, weak anion exchanger. DEAE is also a weak exchanger, the other possibility is MonoQ

  1. Dialyze into buffer C (no NaCl) for ~4hrs
  2. Store at -80

All experiments run in buffer H

Buffers

Buffer A:

  • 20mM monoethanolamine (pH 9.0)
  • 20% glycerol
  • 100mM KCl
  • 5mM MgCl2
  • 1mM AEBSF (see notes)
     Substitude PMSF
  • 1mM DTT
  • 0.1mM ATP

Buffer B:

  • 20mM monoethanolamine (pH 9.0)
  • 10% glycerol
  • 10mM KCl
  • 5mM MgCl2
  • 1mM AEBSF
  • 1mM DTT
  • 2.5% NP40 (see notes)
  • 0.2% DHPC (see notes)


Buffer C:

  • 20mM monoethanolamine (pH 9.0)
  • 10% glycerol
  • 10mM KCl
  • 5mM MgCl2
  • 1mM AEBSF
  • 1mM DTT
  • 0.5% NP40

Buffer H:

  • 50mM Tris-Cl (pH 7.6)
  • 100mM KCl
  • 25uM Zn(CH3COO)2
    (Zinc Acetate)
  • 0.1% NP40
  • 10mM MgCl2
  • 1mM DTT

Notes

  • note- PMSF must be dissolved in isopropanol, and should be used in the hood.
  • NP40 is no longer available, use NP40 substitute.
  • artifical lipid, to reduce strain from conformational change between membrane and micelle
  • Try without PMSF beyond Buffer A and possibly Buffer B.

Wild-type FtsH protein was expressed in E.coli strain JM105 pBHB1. pBHB1 overexpresses FtsH under Arabinose control (Herman et al., 1998). Cultures were grown at 34°C to an OD600 of 0.6 in TB-Mg2+ media, and L-arabinose was add to 0.4%. Cells were harvested by centrifugation 2–3 hr later and resuspended in buffer A. The cells were lysed by French press and centrifuged for 10 min at 4000 rpm. The supernatant was collected and centrifuged again at 40,000 rpm. The pellet, containing the membrane fraction, was solubilized in buffer B followed by another centrifugation at 40,000 rpm. The supernatant was load on an FFQ column and eluted with a linear gradient from 10 mM to 1 M NaCl in buffer C. The fractions containing FtsH were then loaded on a Superose 12 column and eluted with buffer C. FtsH-containing fractions were purified further on a Mono P column. The sample was eluted with a linear gradient from 10 mM to 1 M NaCl in buffer C. Purified FtsH was dialyzed in buffer C for 4 hr and stored at _80°C.

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