Editing Protein Purification from Inclusion Bodies Protocol

== Expression of recombinant proteins<br> ==

'''Materials:'''<br>- LB medium<br>- 40% glucose<br>- Amp 100 mg/ml<br>- IPTG 100 mg/ml in PBS<br>

<br>'''Methods'''<br>1. Inocculate 100 ml LB medium + 1 ml glucose + Amp with 1 fresh colony<br>2. Grow overnight<br>3. Inocculate 1 l LB medium + 10 ml glucose + Amp , with 1 ml preculture<br>4. Grow till OD600 ~ 0.7, take a 1 ml sample<br>5.&nbsp; Prepare fresh IPTG, add 1 ml<br>6. Incubate 3 hours<br>7. Take 1 ml sample for expression control<br>8. Cool culture 15-20â² on ice<br>9. Centrifuge 30â² at 2000 g, 4 oC<br>10. Pellet can be stored at -80 oC<br>

<br>'''Expression QC'''<br>â¢ pellet the 1 ml samples and wash 2x in 1.5 ml cold PBS<br>â¢ add100 ÎŒl dH2O and vortex<br>â¢ add 20 ÎŒl 5x SDS-loading and boil 5â²<br>â¢ run 12 ÎŒl on a SDS-PAGE gel<br>â¢ stain with Coomassie

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== Purification of inclusion bodies<br> ==

<br>Stock solutions<br>â¢ lysozym 50 mg/ml in dH2O (store at -20 C)<br>â¢ DNase 1 mg/ml in 50% glycerol, 75 mM NaCl (store at -20 oC<br>â¢ MgCl2 0.5 M in dH20<br>â¢ Tris-Cl 2.5 M pH 8.0<br>â¢ NaEDTA 0.5 M in 50 mM Tris-Cl, pH 8.0<br>â¢ NaAzide 30% in PBS<br>â¢ Na deoxycholate 10% in dH2O<br>â¢ Triton X-100 10%<br>â¢ 5 M NaCl<br>â¢ DTT 1 M

'''Buffers'''<br>

<br>'''Solution buffer''' (13 ml), pH 8.0<br>50 mM Tris-Cl 260 ÎŒl stock<br>25% sucrose 3.25 g<br>1 mM NaEDTA 26 ÎŒl stock<br>0.1% NaAzide 43.3 ÎŒl stock<br>10 mM DTT 130 ÎŒl stock

'''Lysis buffer''' (12.5 ml)<br>50 mM Tris-Cl 250 ÎŒl stock, pH 8.0<br> 1% Triton X-100 1.25 ml stock<br> 1% Na deoxycholate 1.25 ml stock<br> 100 mM NaCl 250 ÎŒl stock<br> 0.1% NaAzide 42 ÎŒl stock<br> 10 mM DTT 125 ÎŒl stock

'''Washing buffer with Triton''' (10 ml)<br>50 mM Tris-Cl 200 ÎŒl stock<br>pH 8.0 0.5% Triton X-100 0.5 ml stock<br>100 mM NaCl 200 ÎŒl stock<br>1 mM NaEDTA 20 ÎŒl stock<br>0.1% NaAzide 33.3 ÎŒl stock<br>1 mM DTT 10 ÎŒl stock<br>

<br>'''Washing buffer without Triton''' (10 ml)<br>50 mM Tris-Cl 200 ÎŒl stock<br>pH 8.0 100 mM NaCl 200 ÎŒl stock<br>1 mM NaEDTA 20 ÎŒl stock<br>0.1% NaAzide 33.3 ÎŒl stock<br>1 mM DTT 10 ÎŒl stock

11. Resuspend pellet in 13 ml solution buffer on ice and transfer it to a 30 ml centrifugion bottle<br>12. Sonicate 50% level 4-5, 30 pulses, on ice<br>13. Add 100 ÎŒl lysozym, 250 ÎŒl DNase I, 50 ÎŒl MgCl2, vortex<br>14. Add 12.5 ml lysis buffer and vortex short<br>15. Incubate 30-60â² at RT<br>16. Add 350 ÎŒl NaEDTA<br>17. Freeze in N2 until it stops bubbeling and thaw for 30â² at 37 oC<br>18. Add 200 ÎŒl MgCl2<br>19. Wait until viscosity decreases 30-60â²<br>20. Add 350 ml NaEDTA

From now on everything should be done on ice<br><br>

21.&nbsp; Pellet at 11,000 g, 20â², 4 oC<br>22.&nbsp; Discard supernatant and take a sample<br>23. Resuspent pellet in 10 ml washing buffer with Triton<br>24.&nbsp; Sonicate 50% level 4-5, 30 pulses, on ice<br>25. Pellet at 11,000 g, 20â², 4 oC<br>26. Discard supernatant and take a sample<br>27. Resuspent pellet in 10 ml washing buffer without Triton<br>28. Sonicate 50 % level 4-5, 30 pulses, on ice<br>29. Pellet at 11,000 g, 20â², 4 oC<br>30. Discard supernatant and take a sample

== Dissolving of recombinant protein<br> ==

31. Dissolve pellet in 9 ml 8 M Guanidinium pH 8.0 + 4 mM DT<br>32. Shake at RT until pellet dissolves<br>33. Aliquot in 1 ml fractions and store at -80 oC

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== Refolding<br> ==

<br>'''200 ml refolding buffer'''<br>100 mM Tris-Cl: 8 ml stock<br>400 mM L-Arginine: 16.9 g<br>2 mM NaEDTA: 0.8 ml stock<br>0.5 mM ox.glutathione: 61.2 mg<br>5 mM red. glutathione: 308 mg<br>protease inhibitors: 50 ÎŒl cocktail

34. Add 1 ml dissolved protein drop by drop using a 1 ml syringe and a # 27 meedle to the refolding buffer while vigorously stirring.<br>

35. Stir slow, 8 hours at 4 oC<br>

36. Again add 1 ml <br>

37. Stir slow, 8 hours at 4 oC.<br>

38. Again add 1 ml<br>39. Stir slow, 8 hours at 4 oC<br>40. Filter through a 2 um filter and concentrate till ~ 12 ml in a 200 ml concentrator<br>41. Wash concentrator with 1 ml flow through and add this to the protein sample<br>42. Measure OD280, it should be at least 3x higher than flow through<br>

== Size exclusion(FPLC)<br> ==

<br>Elution buffer<br>150 mM NaCl<br>20 mM Tris-Cl pH 8.0 (2um filtered)<br>43. Filter the protein sample through a 0.2 um filter<br>44. Bring 13 ml protein on the size exclusion column (hiload 26/60 Superdex 200 prep grade) using a superloop<br>45. Elute with the elution buffer 1ml/min, start collection 4 ml fractions from 70 ml-300 ml (unfolded protein comes of in the void volume)<br>46. Determine amount of protein in different fractions and run fractions on gel to check the purity of the protein<br><br><br><br>