Protein Purification from Inclusion Bodies Protocol

Expression of recombinant proteins

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

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

Expression QC
• pellet the 1 ml samples and wash 2x in 1.5 ml cold PBS
• add100 ÎŒl dH2O and vortex
• add 20 ÎŒl 5x SDS-loading and boil 5′
• run 12 ÎŒl on a SDS-PAGE gel
• stain with Coomassie

Purification of inclusion bodies

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

Buffers

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

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

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

Washing buffer without Triton (10 ml)
50 mM Tris-Cl 200 ÎŒl stock
pH 8.0 100 mM NaCl 200 ÎŒl stock
1 mM NaEDTA 20 ÎŒl stock
0.1% NaAzide 33.3 ÎŒl stock
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
12. Sonicate 50% level 4-5, 30 pulses, on ice
13. Add 100 ÎŒl lysozym, 250 ÎŒl DNase I, 50 ÎŒl MgCl2, vortex
14. Add 12.5 ml lysis buffer and vortex short
15. Incubate 30-60′ at RT
16. Add 350 ÎŒl NaEDTA
17. Freeze in N2 until it stops bubbeling and thaw for 30′ at 37 oC
18. Add 200 ÎŒl MgCl2
19. Wait until viscosity decreases 30-60′
20. Add 350 ml NaEDTA

From now on everything should be done on ice

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

Dissolving of recombinant protein

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

Refolding

200 ml refolding buffer
100 mM Tris-Cl: 8 ml stock
400 mM L-Arginine: 16.9 g
2 mM NaEDTA: 0.8 ml stock
0.5 mM ox.glutathione: 61.2 mg
5 mM red. glutathione: 308 mg
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.

35. Stir slow, 8 hours at 4 oC

36. Again add 1 ml

37. Stir slow, 8 hours at 4 oC.

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

Size exclusion(FPLC)

Elution buffer
150 mM NaCl
20 mM Tris-Cl pH 8.0 (2um filtered)
43. Filter the protein sample through a 0.2 um filter
44. Bring 13 ml protein on the size exclusion column (hiload 26/60 Superdex 200 prep grade) using a superloop
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)
46. Determine amount of protein in different fractions and run fractions on gel to check the purity of the protein