| Yang Y, Dignam JD, Gentry LE
(1997)
Biochemistry,
36,
11923-11932 |
| Functional Studies |
| N-terminal poly-histidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| HMS174 and BL21 |
| 37.0 |
| 4h |
| pET16b |
| 2ml overnight culture was diluted to 500ml in LB medium and grown with shaking at 37degC. when OD600 reached 0.6-0.8, expression was induced with 1mM IPTG and incubation continued af further 4h. Cells were harvested by centrifugation (5000g, 10min) and suspended in 10ml of 50mM TrisHCl pH 8.0, 1mM EDTA. Cells were then harvested and stored at -70degC. |
| IPTG |
| OD 600 =
0.6-0.8 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| 1:50mM TrisHCl pH 8.0, 1mM EDTA, 0.5% Triton X-100 2:50mM TrisHCl pH 8.0, 1mM EDTA, 2M urea |
| 6M GdnHCl, 400mM NaCl, 50mM potassium phosphate pH 8.0, 10mM 2-mercaptoethanol, 1% Triton X-100, 10% glycerol |
| 1: 50mM TrisHCl pH 8.0, 2mM EDTA, 0.1% PEG3350, 2: 2mM GSH, 0.5mM GSSG, 50mM TrisHCl pH 8.0, 100mM NaCl, 0.2M L-arginineHCl, 2mM EDTA, 0.1% PEG |
| Metal affinity chromatography |
| no |
| 8.0 |
| 4.0 |
| 0.1mg/ml |
| 6days |
| GSH/GSSG |
| 2mM, 0.5mM |
| Frozen cells were thawed on ice, resuspended in 10ml of 50mM TrisHCl pH 8.0, 1mM EDTA and sonicated (3 x 20sec). After centrifugation (5000g, 10min), the pellet was washed three times in 10ml wash buffer 1, then once with 10ml wash buffer 2. The pellet was then washed twice with deionized water and suspended in 5ml solubilization buffer, then mixed gently at room temperature overnight, followed by centrifugation (4degC, 12000g, 30min).
The supernatant was then added to 2ml of Ni-NTA resin equilibrated with solubilization buffer. The resin was washed, then the protein was eluted by step elution with imidazole in solubilization buffer.
The protein was then refolded by a series of dialyses against 100 volumes of buffer. Firstly, the protein was dialyzed against Refolding buffer 1 containing 4.5M GdnHCl for 30min, followed by refolding buffer 1 containing 4.0M GdnHCl for 30min. This 2-step dialysis series was repeated 3 times.
The sample was then dialysed step-wise (30min at each concentration) against 3.75, 3.5, 3.25, 3 and 2M GdnHCl in refolding buffer 1 containing 0.3M Na2SO3, 0.03M Na2S4O6. The dialysis against 2M GdnHCl was over 12-16h, then the buffer was changed to 1M GdnHCl in refolding buffer 1 and dialysis continued for 6h.
The protein was then dialyzed against refolding buffer 2 for 4 days. The buffer was then changed to 50mM TrisHCl pH 8.0, 100mM NaCl and dialysis continued for 12-16h. |
| Bioactivity |
| None |
| L-Arginine,Polyethylene glycol (PEG) |
| .2M0.1% |
| n/a |
| n/a |
| n/a |