Paul.xu at 02:03, 10 August 2014

2014-08-10T02:03:30Z

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== 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 <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  Recombinase ==

Based on Nature Biotechnology

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

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

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

4.  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%  to 1 L--> 10 mM, add 1 ml of 15%--> 1 mM

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

6. Spin down cells. Freeze and thaw with 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 Lysis Buffer.

8. Wash column with  Lysis Buffer without glycerol.

9. Elute with linear gradient of imidazole from 10 mM  imidazole in Lysis Buffer to 300 mM  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 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 FLP Purification Buffer).

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

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

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

16. Wash with 400 mM NaCl in FLP Purification Buffer.

17. Elute protein with linear gradient from 400 mM to 1 M NaCl in 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]]

FLP recombinase Purification - Revision history

Paul.xu at 02:03, 10 August 2014