GST Protein Purification

Materials and Reagents

  • BL21 DE3 cells (Sigma-Aldrich)

  • LB

  • Ampicillin

  • IPTG

  • Lysis buffer (50mM Tris, pH 8.0, 150mM NaCl, 0.1 mM EDTA)

  • Glutathione agarose (Prometheus Protein Biology Products)

  • Elution buffer (50mM Tris, pH 8.0, 150mM NaCl, 0.1 mM EDTA, 10 mM reduced glutathione)

  • TEV protease (Sigma-Aldrich)

Equipment

  • Qsonica Q500 sonicator

  • Nutator

  • Column

Procedure

  1. Transform protein expression plasmids into BL21 DE3 cells (Sigma-Aldrich).

  2. Inoculate starter cultures with one colony and grow overnight in 5mL of LB at 37°C supplemented with ampicillin.

  3. Add the starter culture to 1L of LB with ampicillin.

  4. Grow at 37°C until OD600 was 0.6.

  5. Induce with a final concentration of 100 μM IPTG.

  6. Pellet bacteria and resuspend in lysis buffer (50mM Tris, pH 8.0, 150mM NaCl, 0.1 mM EDTA).

  7. Lyse using Qsonica Q500 sonicator.

  8. Centrifuge at 23000 x g for 20 min.

  9. Add the supernatant to glutathione agarose equilibrated in lysis buffer and incubate on a nutator for 1 h at 4°C.

  10. Centrifuge the agarose at 800 x g for 1 min and wash with lysis buffer 3 times.

  11. Elute protein with elution buffer (50mM Tris, pH 8.0, 150mM NaCl, 0.1 mM EDTA, 10 mM reduced glutathione).

  12. To cleave the GST-tag, incubate protein with TEV protease (Sigma-Aldrich), add to equilibrated glutathione agarose, incubate on a nutator for 1 h at 4°C overnight, place beads on column, and collect flow through as cleaved protein.