Editing FLP recombinase Purification (section)

== Expression and Purification of FLP &nbsp;Recombinase  ==

Based on Nature Biotechnology

1. Transform BL21 cells such as Nico21(DE3)pLysN with plasmid&nbsp;2044 pBad24-Tat-yFLP. 

2. &nbsp;Inoculate 500 ml LB/amp/chl and grow at 37 oC o/n 

3. &nbsp;Add to 8 L LB/amp/chl, grow to O.D600~ 0.5 

4. &nbsp;Cool to 23 oC 

5. Add 10 mM L-arabinose, induce 3~5 hours at 23 oC

Mw of arabinose is 150, 15% arabinose is 1 M, add 10 ml of 15% &nbsp;to 1 L--&gt; 10 mM, add 1 ml of 15%--&gt; 1 mM

Maybe add 1 mM to induce o/n at 23 oC for better solubility

6. Spin down cells. Freeze and thaw with&nbsp;200 ml Lysis Buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 0.1 mM beta-ME, 5% glycerol) 

7. Load to a 10 ml Ni column equilibrated with&nbsp;Lysis Buffer. 

8. Wash column with &nbsp;Lysis Buffer without glycerol. 

9. Elute with linear gradient of imidazole from 10 mM &nbsp;imidazole&nbsp;in Lysis Buffer to 300 mM&nbsp; imidazole in Buffer B (20 mM Tris, pH 7.5, 50 mM NaCl, 0.1 mM beta-ME, 5% glycerol. 

10. Dilute eluted FLP with 5 volume FLP dilution buffer (20 mM Tris, pH 7.5, 0.1 mM beta-ME, 5% glycerol) 

11. Load to P-cellulose column equilibrated&nbsp;FLP Purification Buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 0.1 mM beta-ME, 5% glycerol). 

12. Wash column with linear gradient of NaCl (50 to 500 mM NaCl in&nbsp;FLP Purification Buffer). 

13. Elute &nbsp;protein with 0.75 M NaCl in&nbsp;in FLP Purification Buffer 

14. Dilute eluted protein with equal volume of&nbsp;FLP Purification Buffer. 

15. Load to an Mono-S column or SP HP column equilibrated with&nbsp;FLP Purification Buffer 

16. Wash with 400 mM NaCl in&nbsp;FLP Purification Buffer. 

17. Elute protein with linear gradient from 400 mM to 1 M NaCl in&nbsp;FLP Purification Buffer. FLP will be eluted at 500 mM NaCl. For MonoS column flow rate is 0.7 ml/min. 

18. Pool fractions containing FLP protein and dialyse vs FLP Storage Buffer (20 mM HEPES, pH 7.4, 500 mM NaCl, 1 mM DTT, 50% glycerol). 

19. Measure protein concentration using nanodrop. Run SDS-PAGE to check purity. Record data in protein database

[[Category:Protocols]]