Expression and Purification of GST fusion proteins in BL21 cells

Expresssion of GST Fusions in BL21 cells

1.     Innoculate starter culture (3mL) in LB w/ antibiotic

2.     Dilute starter 1:100 and grow for 2hr 15min at 37C

3.     Induce protein expression by adding 0.1mM IPTG (10,000X lab stock) and transfer culture to cold room shaker set at 16-20C.

4.     After 16h in cold room, pellet bacteria in 50mL tubes by spinning at 4000RPM for 10 minutes.

5.     Discard super and either freeze pellets at –80C or begin lysis.

Purification of GST fusion proteins

Lysis buffer

50mM Tris pH 8

150mM NaCl

0.05% NP40

0.5mM PMSF (add fresh!!!)

Elution buffer

100mM Tris pH 8.0

10% Glycerol

15mg/mL reduced glutathione

SET PH to 8!!! (Using 10N NaOH, adding 7.5uL to 1mL elution should set pH to 8.0)

1      Resuspend bacteria in pre-chilled lysis buffer (~10mL for a pellet from 50-100mL culture)

2      Add 25ug/mL Lysozyme (1:2000) and leave on ice for 0.5h-1h

3      Transfer lysate to 13 mL JA20.1 hard plastic centrifuge tube

4      Sonicate 2X at 18% output for 20s (1s ON / 1s OFF)

5      Spin @ 12.5k RPM for 20 minutes (Make sure balance is within 50mg for opposing tubes)

6      Wash 25uL glutathione beads 3 times with lysis buffer (Spin beads for x400g for 2min)

a.     Use a wide-bore pipette tip when handling beads and never spin >x400g

7      After spinning, keep the supernatant, decanting into a 15mL tube containing pre-washed glutathione beads

8      Incubate >3h at 4C on a rotator

9      After binding, wash beads 3 times with 5mL lysis buffer (5 min rotating @4C each wash)

10   Transfer beads to Eppendorf tube in 1mL lysis buffer

11   Remove residual buffer from beads with a gel loading tip (don’t take the beads though!)

12   Elute by adding 100uL elution buffer – resuspend and rotate for 2hrs @4C

13   Spin down beads and take supernatant (This contains your protein)

14   Analyze 2uL protein via SDS-PAGE // Measure protein conc. using Bradford assay