Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ferric uptake regulation protein |
| Abbreviated Name | Fur |
| SCOP Family | FUR-like |
| Structure Notes | |
| Organism | Pseudomonas aeruginosa |
| UniProt Accession | Q03456 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 134 |
| Molecular Weight | 15234.5 |
| Pi | 5.62 |
| Molecular Weight | 15234.5 |
| Disulphides | Unknown |
| Full Sequence |
MVENSELRKAGLKVTLPRVKILQMLDSAEQRHMSAEDVYKALMEAGEDVGLATVYRVLTQFEAAGLVVRHNFDGGHAVFELADSGHHDHMVCVDTGEVIEFMDAEIEKRQKEIVRERGFELVDHNLVLYVRKKK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lewin AC, Doughty PA, Flegg L, Moore GR, Spiro S. (2002) Microbiology, 148, 2449-2456 |
| Project Aim | Identification and Characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pET21 |
| Expression Protocol | Fur was purified from the pET21 clone in BL21(DE3) initially using the method of Ochsner et al. (1995) . Subsequently a modified procedure was developed, which did not involve metal-affinity chromatography. Cultures (500 ml) were grown to an OD650 of 0·5–0·6, induced with 1 mM IPTG, then incubated overnight. Cells were harvested, washed in 50 mM Tris/HCl (pH 7·9)-0·5 mM EDTA-50 mM NaCl, then resuspended in the same buffer containing 1 mM PMSF and 1 mM DTT. After sonication, the cell-free extract was applied to a 300 ml DEAE cellulose ion-exchange column equilibrated with 50 mM Tris/HCl (pH 7·9)-50 mM NaCl, and then eluted with a linear 50–500 mM NaCl gradient in the same buffer. Fur-containing fractions (as judged by SDS-PAGE) were dialysed overnight in 20 mM Tris/HCl (pH 7·0) then applied to a 20 ml heparin-agarose column equilibrated with 20 mM Tris/HCl (pH 7·0). The column was washed with the same buffer, then Fur was eluted with 20 mM Tris/HCl (pH 7·0)-1 M NaCl. For purification of the MBP fusion, 500 ml cultures were grown in Lennox broth containing 0·2% (w/v) glucose to an OD650 of 0·5–0·6, induced with 1 mM IPTG, then incubated overnight. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM Tris/HCl (pH 7·9)-100 mM NaCl |
| Solubilization Buffer | 3 M guanidinium hydrochloride (Gdn.HCl) |
| Refolding Buffer | 20 mM Tris/HCl (pH 7·0)-50 mM NaCl. |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Cells were harvested, washed in 50 mM Tris/HCl (pH 7·9)-50 mM NaCl, then resuspended in the same buffer containing 0·5 mM EDTA, 1 mM PMSF and 1 mM DTT. Cells were disrupted by sonication, clarified, and the cell extract applied to a 15 ml amylose column equilibrated with 50 mM Tris/HCl (pH 7·9)-100 mM NaCl. The column was washed with the same buffer, then the fusion protein eluted with 50 mM Tris/HCl (pH 7·9)-100 mM NaCl-10 mM maltose. The fusion was treated with factor Xa, and then 3 M guanidinium hydrochloride (Gdn.HCl). The partially denatured Fur was purified by FPLC on an S75 gel-filtration column, then renatured by dialysis against 20 mM Tris/HCl (pH 7·0)-50 mM NaCl. |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The refolding temperature is not stated |