Editing Enterokinase Purification (section)

= Old protocol<br> =

<br>

<br>

1. Transform BL21 Star (DE3)/pRare2pLysS with plasmid<br> 

2. Grow cells o/n in 100 ml LB/Kan/Chl.<br> 

3. Inoculate 2 L LB/Kan/Chl with 100 ml (1&nbsp;: 40, v/v) from an o/n preculture.<br> 

4. Grow at 37 °C and 200 rpm to OD600 1.6.<br> 

5. Induce with 0.1 mM IPTG and grow o/n at 30 to 37 oC.<br> 

6. Harvest cells by spining at 6000 rpm.<br> 

7. Resuspend cells with 10 ml of '''lysis buffer''' per gram of wet cell. <br> 

&nbsp;50 mM Tris-HCl, pH 7.0, 2&nbsp;% Triton X-100, 500 mM NaCl<br> 

8. Lysis by freeze-thaw.<br> 

9. Homogenize. <br> 

10. Spin at 12000 rpm for 20 min.<br> 

12. Dissolve pellet&nbsp; in Buffer P: 1g pellet in 20 ml buffer 

7.5 M guanidine-HCl, 50 mm Tris-HCl, 100 mm β-mercaptoethanol.<br> 

13. Adjust pH to 3.5–4,<br>14. Dialyze the solution against 5 mM citrate pH 3.5–4.0.<br> 

15. Spin at 12000 rpm.<br> 

16. Dissolve precipitated protein in 7.5 M guanidine-HCl, 50 mM Tris pH 4.0–4.5.<br> 

17. Load to Ni column and elute with 300 mM imidazole.<br> 

18. Concentrate the protein using spin columns. 

19. Refolding:&nbsp; dropwise dilution of the protein solution into the 100-fold volume<br>of a buffer containing 0.5 M arginine, 50 mM Tris pH 8.3, 20 mM CaCl2 , 1 mM EDTA, 5 mM cysteine, 0.5 mM cystin. <br> 

20. Incubate at 16&nbsp; °C for 72–96 h.<br> 

21. Optional: load the enterokinase concentrate onto a benzamidine sepharose 4 Fast Flow (high sub) column (GE Healthcare). After washing the resin with 50 mm Tris pH 7.5, 500 mm NaCl, correctly folded enterokinase can be eluted with the same buffer supplemented with 25–100 mm benzamidine. <br> 

21. Concentrate 20 times of the original volume using 9kDa spin columns<br> 

22. Load to a TSK gel filtration column and purify using 50 mm Tris pH 7.5, 50 mm NaCl.<br>23. Concentrate the eluates to approximately 0.2 mg/ml, add equal volume of 20&nbsp;% (v/v) glycerol. 

24. Store aliquoted protein at −80 °C<br><br><br> 

[[Category:Protocols]]