| Aruna B, Ghosh S, Singh AK, Mande SC, Srinivas V, Chauhan R, Ehtesham NZ
(2003)
Biochemistry,
42,
10554-10559 |
| Structural Studies |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| M15 |
| 37.0 |
| not stated |
| pQE30 |
| Cells were grown at 37degC in Terrific broth containing kanamycin and ampicillin. Expression was induced with 1mM IPTG. |
| IPTG |
| OD n/a =
n/a |
| Sonication |
| Lysozyme |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| n/a |
| 50mM sodium phosphate pH 7.0, 300mM NaCl, 8M urea |
| 10mM TrisHCl pH 8.0 |
| Metal affinity chromatography |
| no |
| 8.0 |
| 4.0 |
| n/a |
| 9h |
| None |
| n/a,n/a,n/a |
| 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 a Talon column and the protein was purified under denaturing conditions, being eluted in solubilization buffer with 150mM imidazole. Selected fractions were pooled and dialysed against refolding buffer, which was changed three times over 9h. |
| Far-UV Circular Dichroism,Fluorescence,SDS-PAGE,Gel filtration chromatography,Mass spectrometry,Free sulfhydryl group estimation |
| None |
| None |
| n/a |
| n/a |
| >95% |
| n/a |