Paul.xu at 22:40, 18 April 2013

2013-04-18T22:40:13Z

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== Protocol 1, for protein kinases ==

=== Screening: ===

1. Subclone protein kinases from Harvard set into 1517 pT7A3-MBP-(3C)-sfGFP using Sal1 + HindIII. 

2.  Transform Rosetta2 BL21(DE3), or BL21Star/pRare2pLysS with plasmid, grow on LB/Kan/Chl plate 

3.  Screen expression using 2 ml LB/Kan/Chl (glass tubes). Induce with <strike>0.3 mM IPTG at 37 oC for 4 hours</strike>,  or 0.1 mM IPTG o/n at 25 oC. 

4. Purify protein with 20~30 ul beads, run on SDS-PAGE. 

5. Scan gels and document result as detail as possible. 

 

=== Large Scale Expression ===

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

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

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

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

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

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

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

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

9. Spin at 12000 rpm for 20 min at 4 oC. 

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

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. 

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. 

13. Collect supernatant and discard pellet. Wash tubes carefully. Wash rotor with ddH2O. 

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

15. Collect flowthrough  and save a sample for SDS-PAGE analysis. 

16. Wash column with 20 column volumes of wash buffer. 

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. 

18. Measure OD280 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). 

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  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). 

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

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

24. Elute bound protein with Elution Buffer. Collect eluate. 

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

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  5 ~ 10 ml. 

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) 

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

29. Aanalyze peak fractions by nanodrop and SDS–PAGE. Pool fraction containing desired protein.  

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) 

 

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

 

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

[[Category:Protocols]]

High Throughput Protein Production - Revision history

Paul.xu at 22:40, 18 April 2013