Editing FLP recombinase Purification

== FLP without any fusion tag  ==

T7 Promoter: Induction at 37 oC will get insoluble FLP 

Tac promoter: FLp is soluble when induction at 37 oC 

1. inoculate 8 L volume of LB Brath (10g Tryptone. 5 g yeast extract. 5 g NaCl, 0.1 ml of 10 N NaOH per L)/Kan/Chl with a 100 ml of overnight culture of Bl21 strain transformed with pT7A3-iFLP at 37 oC. 

2. Grow cells at 37 oC to OD600=0.7 

3. Cool to 22 oC. 

4. Add IPTG to 50 uM 

5. Grow at 22 oC for 6 hours to o/n 

6. Spin down cells, 

the following steps should be carried out at 4 oC. 

7. Lyse cells in lysis buffer (150mM Tris-HC1, pH 7.4, 50mM (NH4)2SO4, 1mM EDTR, 0.5mM DTT) 

8. Homogenize and then Spin at 25000 rpm for 30 min. 

9. Load protein to a Bio-Rex 70 column (30 ml) equilibrated with BR70 Buffer ( 50 mM NaP04 buffer, pH 6.5. 1 mM EDTA. 0.5 mM DTT, 5% glycerol). 

10. Wash column with 60~100 ml of the same BR70 Buffer 

11. Elute protein with 60 to 100 ml of elution buffer (BR70 Buffer + 700 mM NaCl) 

12. Pool frations containing FLP. 

13. Add solid ammonium sulfate to 60% saturation (add 39 g to 100 ml), stir at 4 oC for 30 min. 

14. Spin at 25000 rpm for 20 min 

15. Dissolve pellet in &lt;5 ml Gel Filtration Buffer (50 mM NaP04 buffer, pH 6.7. 1mH EDTA. 1mM DTT, 5% glycerol and 150 mM NaC1). 

16. Load to a gel filtration column. 

17. Pool fractions containing FLP and load to Mono-S HR 5/5, 5X50 mm column, equilibrated with Gel Filtration Buffer. Flow rate: 0.5 ml/min 

18. Elute protein with 32.6 ml NaCl gradient from 0 to 700 mM in Gel Filtration Buffer. 

19. Pool fractions containing FLP. 

20. Dialyse vs dialysis buffer containing 50% glycerol. 



== 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]]