RNase A Expression

1. Transform  BL21(DE3) with RNase A plasmid.

2. Grow in 200 ml TB medium o/n.

3. Inoculate 7 L, grow at 37 oC.

4. Add IPTG to 2 mM induce for 4 hours  at 37°C.

5. Spin down cells.

6. Resupsend cell pellet in 250 ml of cell lysis buffer (20 mM Tris-HCl buffer, pH 7.8, 6M urea and 1 mM EDTA, shaking for 20 min at 37°C.

7. Spin for 15 min at 30,000 g,

8. Resuspend pellet in 250 ml of solubilization buffer (20 mM Tris-HCI buffer, pH 7.8, 6 M urea,  0.4 M NaCl, 20 mM DTT, 1 mM EDTA

9. Shake the suspension  for 20 min at 37°C.

10. Spin  for 15 min at 30000 g.

11. Collect the supernatant and the add reduced DIT (0.22 g).

12. Stir the supernatant for 10 min at room temperature.

13. Add glacial acetic acid to adjust  the pH to 5.0.

14. Dialyse the resulting solution against 20 mM Tris-acetic acid (AcOH) buffer, pH 5.0, 0.1 M NaCI

15. Remove insoluble material by centrifugation.

16. Oxidize supernatant by dialysising (>24 h) against refolding buffer, 50 mM Tris-AcOH buffer, pH 7.8, 0.1 M NaCI, 1 mM reduced glutathione and 0.2 M oxidized glutathione.

17. Dialyse the refolded RNase A against 20 mM Tris-AcOH buffer, pH 8.0.

18. Load the dialyzed sample to  a column (15.0 cm x 4.9 cm² ) of DE52 anion exchange resin equilibrated with the same buffer.

19. Load the flow-through onto a column (15.0 cmX 1.8 cm²) of S-Sepharose cation exchange resin equilibrated with 20 mM Tris-AcOH buffer, pH 8.0.

20. Wash with 100 ml  of 20 mM Tris-AcOH buffer, pH 8.0.

21. Elute with a linear gradient of NaCl (0.00-0.35 M), in Tris-AcOH buffer, pH 8.0.