| Fan X, Xu D, Lu B, Xia J, Wei D.
(2008)
Journal of biochemical and biophysical methods,
1,
1 |
| Protein refolding |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| None |
| 0.0 |
| 30 |
| unspecifed |
| A 100-g amount of NTA cell paste expressed as inclusion bodies in Escherichia coli was put in 0.1 mol/l Tris–HCl (pH 8.5) containing 2 mmol/l EDTA, 0.2 mg/ml Lysozyme and 2% Triton X-100 and crashed by ultrasonic processor in an ice-water bath and then centrifuged at 12,000 rpm for 30 min. The isolated inclusion body was washed twice with 0.01 mol/l PBS (pH 7.5) containing 0.1 mol/l NaCl and 0.1 mol/l Tris–HCl (pH 8.5) containing1 mmol/l EDTA, respectively. Then, the clean inclusion body was dissolved in 0.1 mol/l Tris–HCl (pH 8.5) containing 8 mol/l urea and 0.2 mol/l DTT. After incubation at 4 °C for 24 h with full agitation, the extract of the NTA was obtained by centrifuging at 12,000 rpm for 30 min. |
| Not Stated |
| OD n/a =
n/a |
| ultrasonication |
| Lysozyme |
| None |
| insoluble |
| Column refolding: Size-exclusion chromatography |
| 0.01 mol/l PBS (pH 7.5) containing 0.1 mol/l NaCl and 0.1 mol/l Tris–HCl (pH 8.5) containing1 mmol/l EDTA |
| 0.1 mol/l Tris–HCl (pH 8.5) containing 8 mol/l urea and 0.2 mol/l DTT |
| mixed buffer B-buffer A (25:75),Buffer A: 0.1 mol/l Tris–HCl (pH 8.5), 1 mmol/l ETDA, 0.8 mol/l arginine, the ratio of GSH to GSSG was 0.9/0.18 mmol/l buffer B: 0.1 mol/l Tris–HCl (pH 8.5), 1 mmol/l ETDA, 8 mol/l urea, the ratio of GSH to GSSG was 0.9/0.1 |
| None |
| no tag |
| 8.5 |
| 0.0 |
| n/a |
| n/a |
| GSH/GSSG |
| 0.9/0.18 mmol/l,0.9/0.18 mmol/l |
| Buffer A: 0.1 mol/l Tris–HCl (pH 8.5), 1 mmol/l ETDA, 0.8 mol/l arginine, the ratio of GSH to GSSG was 0.9/0.18 mmol/l buffer B: 0.1 mol/l Tris–HCl (pH 8.5), 1 mmol/l ETDA, 8 mol/l urea, the ratio of GSH to GSSG was 0.9/0.18 mmol/l. Size-exclusion chromatography refolding was performed using a XK 16/70 column packed with Sepharcryl S-200 gel media. Column was equilibrated with a mixed buffer B-buffer A (25:75). Then, gradually increasing buffer B up to 100%, urea gradient and arginine gradient were formed (as shown in Fig. 1, dark colors represent higher urea concentrations and lower arginine concentrations; light colors represent lower urea concentrations and higher arginine concentrations). Denatured protein of various concentrations were applied to the column and eluted with buffer B as the sample volume was 2 ml. |
| Protein activity assay |
| None |
| None,L-Arginine |
| 0.8 mol/l |
| n/a |
| n/a |
| Note the refolding temperature is not stated in paper |