| Column refolding: hydrophobic interaction chromatography |
| n/a |
| 6 M guanidine hydrochloride (Gdn-HCl), 100 mM Tris–HCl, 1 mM EDTA, 150 mM dithiothreitol (DTT), pH 8.5 |
| C: 100 mM Tris–HCl, 1 mM EDTA, 0.4 M (NH4 )2 SO4 , 0.01% (v/v)B-mercaptoethanol ((B-ME), pH 8.5, 50% v/v glycerol |
| None |
| no tag |
| 8.5 |
| 0.0 |
| n/a |
| n/a |
| Beta-mercaptoethanol |
| 0.01%v/v |
| Refolding by adsorption–elution way in HIC column One hundred microlitres unfolded lysozyme of 10 mg/ml was applied to the Poros PE HIC column (128 mm × 10 mm i.d.) equilibrated with the buffer of high salt concentration (A: 3.6 M (NH4 )2 SO4 , 50 mM Tris–HCl, 1 mM EDTA, 3 mM GSH, 0.3 mM GSSG, pH 8.5) followed by 2 ml gradient of A to the denaturant (B: 8 M urea, 50 mM Tris–HCl, 1 mM EDTA, 3 mM GSH, 0.3 mM GSSG, pH 8.5) and subsequent 1 column volume (CV) washing with A resulting in adsorption as well as prevention of aggregation. After refolding buffer (C: 100 mM Tris–HCl, 1 mM EDTA, 0.4 M (NH4 )2 SO4 , 0.01% (v/v) B-mercaptoethanol ((B-ME), pH 8.5) continuously washed the column to decrease ionic strength, 4 ml gradient of increasing urea concentration from C to B and 4 ml gradient of decreasing urea concentration from B to C in turn was then introduced into the column to release bound lysozyme followed by elution with C to promote released protein refolding. Flow rate was 0.5 ml/min. All steps were carried out on AKTA purifier. In the other experiments, 50% (v/v) of final glycerol concentration was added into the buffer C and then the steps described above were performed. The elution process by the gradient of urea and glycerol in the column and mobile phase is modified according to reference [17], as shown in Fig. 1. As a control experiment, glycerol was immediately mixed with pooled refolded lysozyme. |
| enzyme activity,Bradford assay,SEC-HPLC |
| None |
| Glycerol,(NH4)2SO4 |
| 0.4 M, 50%v/v |
| 86.3% |
| n/a |
| The refolding temperature is not stated |