Editing High Throughput Protein Production (section)

=== Large Scale Expression<br>  ===

1. Grow E coli in 10 ml in LB/Kan/Chl medium at 37 oC o/n <br> 

2. Inoculate 1 ~ 4 L LB/Kan/Chl with 2~ 5 ml culture per L of medium.<br> 

3. When OD600 is ~ 1.2, lower temperature to 25 oC, induce with 0.1 mM IPTG o/n.<br> 

4. Spin down cells, combine pellet and weigh on balance. Record wet weight.<br> 

5. Store pellet at -20 oC or imediately do freeze-thawing for 3 times<br> 

6. Add 15 ml of '''Lysis Buffer''' (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5% glycerol, 10 mM imidazole,&nbsp; 0.1% Triton X-100,&nbsp; 1 mM b-ME) to 1 g of pellet.<br> 

7. Freeze (with dry ice) &amp; thaw 3 times. 

8. Homogenize to broke DNA. Transfer homogenate to 50 ml centrifuge tubes. Cool down centrifuge rotor with dry ice.<br> 

9. Spin at 12000 rpm for 20 min at 4 oC. <br> 

10. Collect supernatant. Add 10 ml lysis buffer to 1 g pellet, homogenize a few seconds.<br> 

11. Spin at 12000 rpm for 20 min at 4 oC. Collect and combine supernatant. Transfer to ultracentrifuge tubes. Balance the tubes carefully. Tighten the caps.<br> 

12. Cool down centrifuge rotor with dry ice. Load tubes to rotor and tighten the lid. Spin at 25000 rpm for 20 min, 4 oC.<br> 

13. Collect supernatant and discard pellet. Wash tubes carefully. Wash rotor with ddH<sub>2</sub>O.<br> 

14. Load supernatant to a HisTrap FF 5 ml columns&nbsp; equilibrated with '''Wash Buffer''' (20 mM Tris-HCl, 500 mM NaCl, 5% glycerol, 30 mM imidazole,&nbsp; 1 mM beta-ME pH 8.0). <br> 

15. Collect flowthrough&nbsp; and save a sample for SDS-PAGE analysis.<br> 

16. Wash column with 20 column volumes of wash buffer.<br> 

17. Elute protein with 5 column volumes of '''Elution Buffer''' (20 mM Tris-HCl, 300 mM NaCl, 5% glycerol, 1 mM b-ME, 250 mM imidazole), collect in 3 ml fractions. <br> 

18. Measure OD<sub>280</sub> using elution buffer as blank. Pool fractions containing protein. 

19. Add 3C protease to the protein solution at a ratio of 1:50 (w:w).<br> 

20. Dialyze overnight (generally 18 h) at 4 oC in '''Dialysis Buffer''' (20 mM Tris-HCl, 150 mM NaCl, 5% glycerol, 1 mM b-ME, pH 8.0). 

21. Load dialysed sample&nbsp; onto a gravity-flow column (Econo-Pac Chromatography Columns, Bio-Rad, Hercules, California, USA) packed with pre-equilibrated Ni Sepharose (2.5 or 5 ml depending on the protein yield).<br> 

22. Very important: '''collect flowthrough, this FT contains purified protein'''.<br> 

23. Wash column with 2 CV of Dialysis Buffer. Collect wash. <br> 

24. Elute bound protein with Elution Buffer. Collect eluate.<br> 

25. Run SDS-PAGE gel on samples from various steps.<br> 

26. Concentrate protein using Pierce 9k protein cencentrator (20 ml) or other 10 K protein concentrator such as Amicon Ultra-15 Centrifugal Filter Units, Concentrator to approximately&nbsp; 5 ~ 10 ml.<br> 

27. Load 5 ml concentrated protein solution to a size-exclusion column (BioRad Biosil TSK 250), prequilibrated with '''SEC Buffer''' (20 mM Tris-HCl, 150 mM NaCl, 5% glycerol, 1 mM b-ME, pH 8.0)<br> 

28. Elute with SEC Buffer and collect 2.5 ml per fraction.<br> 

29. Aanalyze peak fractions by nanodrop and SDS–PAGE. Pool fraction containing desired protein.&nbsp;<br> 

30. Concentrate protein using Pierce 9k protein cencentrator (20 ml) to ~5 ml, measure concentration and dilute with '''Protein Storage Buffer''' (20 mM Tris-HCl, 150 mM NaCl, 50% glycerol, 1 mM DTT, pH 7.5)<br> 

<br> 

Modified from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169394/<br> 

<br> 

If the target protein is a basic protein (pI &gt;8), use Phosphate buffers (pH 7.0 ~ 7.4), do not use Tris Buffer.

[[Category:Protocols]]