| Raghu P, Ghosh S, Soundarya K, Haseeb A, Aruna B, Ehtesham NZ
(2004)
Biochemical and Biophysical Research Com,
313,
642-646 |
| Structural Studies |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| M15 |
| 27.0 |
| 12h |
| pQE30 |
| Cells were grown at 37degC in Terrific broth containing kanamycin and ampicillin. Expression was induced with 1mM IPTG and cells were grown at 27degC for a further 12h. |
| IPTG |
| OD n/a =
n/a |
| Sonication |
| Lysozyme |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| n/a |
| 25mM TrisHCl pH 8.0, 8M GdnHCl |
| 25mM TrisHCl pH 8.0, 0.15M NaCl, 5mM imidazole, 10mM glutathione, 0.7M L-arginineHCl, 1% D-glucose |
| Metal affinity chromatography |
| no |
| 8.0 |
| 25.0 |
| n/a |
| n/a |
| GSH |
| 10mM,10mM,10mM |
| Harvested cells were suspended in 50mM sodium phosphate pH 7.0, 300mM NaCl containing 0.1mg/ml lysozyme and incubated at 4degC for 1h. The cells were then lysed by sonication at 4degC for 10min. After centrifugation (13000rpm, 30min), the inclusion bodies were dissolved in solubilization buffer and centrifuged again. The supernatant was loaded onto an Ni-NTA column equilibrated with 25mM TrisHCl pH 8.0, 0.15M NaCl (TBS) with 8M GdnHCl, 1mM DTT. After washing, the protein was subjected to on-column refolding by a gradient of TBS containing 6M GdnHCl and refolding buffer. The protein was eluted in refolding buffer containing 500mM imidazole. Selected fractions were then dialyzed against TBS at 4degC. |
| Far-UV Circular Dichroism,SDS-PAGE,Gel filtration chromatography |
| None |
| L-Arginine |
| 0.7M |
| n/a |
| n/a |
| n/a |