Refolding Record:
| Protein | |
|---|---|
| Protein Name | Luciferase |
| Abbreviated Name | n/a |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Photinus pyralis (North American firefly) |
| UniProt Accession | P08659 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 551 |
| Molecular Weight | 60745.2 |
| Pi | 6.41 |
| Molecular Weight | 60745.2 |
| Disulphides | Unknown |
| Full Sequence |
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sharma SK, Goloubinoff P, Christen P. (2008) Biochemical and Biophysical Research Com, 1, 1 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution/ chaperone-assisted refolding |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidine hydrochloride, 50 mM Tris acetate, 5 mM TCEP (Tris[2-carboxyethyl]phosphine, a non-thiol reducing agent), pH 7.5 |
| Refolding Buffer | 50 mM Tris acetate, 100 mM potassium perchlorate, 15 mM magnesium acetate, pH 7.5, containing the indicated concentrations of Cd2+, 3.5 lM DnaK, |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | Luciferase (17.5 lM) was chemically denatured in 6 M guanidine hydrochloride, 50 mM Tris acetate, 5 mM TCEP (Tris[2- rboxyethyl]phosphine, a non-thiol reducing agent), pH 7.5, for 30 min at 25 °C. Spontaneous, unassisted refolding at 25 °C was initiated through 1:50 dilution (final concentration of luciferase 350 nM) with refolding buffer (50 mM Tris acetate, 100 mM potassium perchlorate, 15 mM magnesium acetate, pH 7.5), containing the indicated concentrations of Cd2+. Luciferase activity was measured in samples of the refolding solution at the indicated times. Error bars represent the SEM from three independent experiments. (B) Chaperone-assisted refolding of chemically denatured luciferase in the presence of 3.5 lM DnaK, 0.7 lM DnaJ, 1.4 lM GrpE and 5 mM ATP under otherwise identical conditions as in Experiment A. |
| Refolding Assay | enzyme activity |
| Refolding Chaperones | DnaK,DnaJ,GrpE |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |