Inclusion body purification

Grow bacteria overnight in -80 oC freezer

Homogenize with buffer A (optional)

Harvest  by centrifugation (optional)

Resuspend in 50 mM NaH2PO4, 150 mM NaCl, 10 mM MgCl2, 0.25% Triton X-100, pH 7.2

Digest nucleic acids   with 1500 U Benzonase (Sigma-Aldrich) for 1 h  at room temperature

Harvest inclusion bodies containing recombinant protein by centrifugation (optional)

Three washes with 100 mM Tris-Cl, 5 mM EDTA, 5 mM DTT, 2% Triton X-100, pH 7.0

Solubilize inclusion bodies in 8 M urea, 100 mM Tris-Cl, 100 mM DTT, pH 8.0

If not fully dissolved, use filter and/or biorad to filter out the supernatant

Load on a Ni-NTA column (Qiagen), which has been previously equilibrated with 8 M Urea, 100 mM NaH2PO4, 10 mM Tris-Cl, 2 mM β-mercaptoethanol, pH 8.0.

Elute bound protein   with 8 M urea, 100 mM NaH2PO4, 10 mM Tris, 2 mM β-Mercaptoethanol pH 4.5

Dialyse against 2 M urea, 50 mM NaH2PO4, 0.5 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 10% glycerol, pH 8.5.

Refold protein  by stepwise dialysis against

50 mM NaH2PO4, 0.5 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 10% glycerol, pH 8.5,

then against 50 mM NaH2PO4, 10% glycerol, pH 8.5.