Preparation of GST fusion proteins

Reference: Smith and Johnson, Gene 67, 31-40 (1989); Smith et al., PNAS 83:8703.
Date of this version: 11/4/96 (by Genhong Cheng)

Reagents:

#1. Growth medium
LB ampicillin (100 µg/ml)

#2. 1000x IPTG
100 mM in distilled water (0.238g in 10 ml H2O), stored at -20 C.

#3. lysis buffer

Reagent [final] stock Volume

EDTA pH 8.0 100 mM 0.5 M 10 ml
Triton X-100 1% 100% 0.5 ml
PBS 1x 1x 38.5 ml
PMSF 1mM 100 mM* 0.5 ml
Aprotinin 1% 100%** 0.5 ml

*100 mM PMSF, 0.174 g in 10 ml of Isopropanol, add just before use
**aprotinin stock solution from Sigma; add just before use

#4. glutathione-agarose obtained from Sigma

#5. PBS

#6. Glutathione (reduced): Make fresh solution of 20 mM in 50 mM tris pH 8.0

Procedure

• Grow a 50 ml overnight culture in LB of bacteria with the desired GEX construct.
• Dilute the overnight (1:20) into 1000 ml fresh LB+ and grow at 37 C for 2 hours.
  Add 2ml ml 100mM IPTG ( final concentration of 100 µM).
• Grow another 2-4 hours after addition of IPTG
• Spin down bacteria (JLA, 4000 rpm x 10 min, 4 C).
• Drain medium out of bottles as complete as possible.
• Resuspend the pellets in 15 ml of lysis buffer and transfer the bacteria into 50 ml tube.
  Sonicate 1 minutes at the setting of 4 (keep on ice during sonication; avoid foaming).
• Pellet membranes from lysed bacteria (JA-12, 4000 rpm x 10 minutes, 4 C ).
• Place supernatant in 50 ml conical screwcap tubes
• Add 2 ml glutathione beads
• Rock gently at 4 C for 1 to 3 hours.
• Spin down the beads in TJ6, set 5 for 5 minutes, 4 C
• Suck off the supernatants.
• Add 10 ml Lysis buffer and resuspend the beads.
• Spin down the beads in TJ6, set 5 for 5 minutes, 4 C
• Suck off the supernatants.
• Add 10 ml PBS with and resuspend the beads.
• Spin down the beads in TJ6, set 5 for 5 minutes, 4 C
• Suck off the supernatants.
• Add 10 ml PBS with and resuspend the beads.
• Spin down the beads in TJ6, set 5 for 5 minutes, 4 C
• Suck off the supernatants.
• Add 10 ml PBS with and resuspend the beads.
• Spin down the beads in TJ6, set 5 for 5 minutes, 4 C
• Suck off the supernatants.
• Add 10 ml PBS with and resuspend the beads.
• Pour beads into small column in cold room.
• Add 3 ml of 20 mM glutathione.
• Collect 0.5 ml for each fraction.
• Take 1 ul from each fraction and check protein concentration
• Combine two fractions that have highest protein concentrations.
• Store GST fusion proteins in 4 C.