| Kim BJ, Mangala SL, Hayashi K.
(2005)
FEBS Letters,
579,
3075-80 |
| Protein refolding |
| N-terminal +C terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 0.0 |
| 00 |
| pET28b |
| Recombinant N- and C-terminal peptide fragments, consisting of a tag of six histidine residues at the N-terminus of N-terminal and six at the C-terminus of C-terminal fragments, were produced as inclusion bodies, respectively. Inclusion body pellets were solubilized in an 8 M urea solution containing 50 mM Tris/HCl (pH 8.0), 1 mM ethylene diamine tetraacetic acid (EDTA) and reduced for 1 h at room temperature by adding 0.05% (v/v) 2-mercaptoethanol and then oxidized by the addition of glutathione (oxidized form) at a concentration of 0.08% (w/v) for 1 h. The insoluble portion was removed by centrifugation at 15 000 × g for 20 min. The denatured fragments were purified using metal chelate chromatography followed by ion-exchange chromatography. The purity of the eluted fractions was assessed with 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). |
| Not Stated |
| OD n/a =
n/a |
| Not stated |
| None |
| Metal affinity chromatography/ion exchange chromatography |
| insoluble |
| Dialysis |
| n/a |
| 8 M urea solution containing 50 mM Tris/HCl (pH 8.0), 1 mM ethylene diamine tetraacetic acid (EDTA) |
| Acetate (pH 5.0), Tris/HCl (pH 8.0) or 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) (pH 10.0), containing 0.16% oxidized glutathione and 8 M urea |
| Metal affinity chromatography/ion exchange chromatography |
| no |
| 8.0 |
| 4.0 |
| n/a |
| n/a |
| None |
| n/a |
| Each of purified N- or C-terminal peptide fragments was subjected to refolding by slow dialysis at 4 °C against 50 mM of different buffers, Acetate (pH 5.0), Tris/HCl (pH 8.0) or 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) (pH 10.0), containing 0.16% oxidized glutathione and 8 M urea. The urea concentration was decreased from 8 to 0 M over 4 days [8]. Enzyme activity during refolding was periodically checked by mixing the two refolding fractions of N- and C-terminal peptide fragments. Co-refolding was also carried out by the same method of refolding, except that the two fragments of N- and C-terminal peptide were mixed prior to the refolding. Enzyme activity in the co-refolded solution was determined periodically. Native and two co-refolded enzymes with catalytic activity (N421/422C and N466/422C) were further purified by ion exchange chromatography. Purity was confirmed by SDS–PAGE analysis. |
| Enzyme activity,Kinetic analysis |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |