| 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 min |
| 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 |
| Dilution |
| 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 |
| 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 |
| None |
| no tag |
| 8.5 |
| 4.0 |
| n/a |
| 24 h |
| GSH/GSSG |
| 0.9/0.18 mmol/l |
| Refolding by dilution
Denatured proteins with various concentrations were rapidly diluted into renaturation buffer: 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. The dilution factor (a dilution factor of 55) was as same as the gel filtration refolding process. After incubation at 4 °C for 24 h, the protein activity was determined. |
| Protein activity assay |
| None |
| L-Arginine |
| 0.8 mol/l |
| n/a |
| n/a |
| n/a |