Refolding Record:
| Protein | |
|---|---|
| Protein Name | Luciferase (Luciferin 4-monooxygenase) |
| Abbreviated Name | Lusiferase |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Firefly (Luciola lateralis) |
| UniProt Accession | Q01158 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 548 |
| Molecular Weight | 60125.4 |
| Pi | 6.5 |
| Molecular Weight | 60125.4 |
| Disulphides | Unknown |
| Full Sequence |
MENMENDENIVYGPEPFYPIEEGSAGAQLRKYMDRYAKLGAIAFTNALTGVDYTYAEYLEKSCCLGEALKNYGLVVDGRIALCSENCEEFFIPVLAGLFIGVGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVITVQKTVTAIKTIVILDSKVDYRGYQSMDNFIKKNTPQGFKGSSFKTVEVNRKEQVALIMNSSGSTGLPKGVQLTHENAVTRFSHARDPIYGNQVSPGTAILTVVPFHHGFGMFTTLGYLTCGFRIVMLTKFDEETFLKTLQDYKCSSVILVPTLFAILNRSELLDKYDLSNLVEIASGGAPLSKEIGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDKPGASGKVVPLFKAKVIDLDTKKTLGPNRRGEVCVKGPMLMKGYVDNPEATREIIDEEGWLHTGDIGYYDEEKHFFIVDRLKSLIKYKGYQVPPAELESVLLQHPNIFDAGVAGVPDPIAGELPGAVVVLEKGKSMTEKEVMDYVASQVSNAKRLRGGVRFVDEVPKGLTGKIDGKAIREILKKPVAKM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Minami Y, Kawasaki H, Minami M, Tanahashi N, Tanaka K, Yahara I. (2000) Biologycal Chemistry, 275, 9055-9061 |
| Project Aim | Protein refolding |
| 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 |
| Wash Buffer | n/a |
| Solubilization Buffer | thermally inactivated for 5 min at 45 or 50 °C, or for 10 min at 42 °C at a final concentration of 0.26 uM in 30 mM MOPS-KOH, pH 7.2, 2 mM dithiothrei |
| Refolding Buffer | 10 mM MOPS-KOH, pH 7.2, 50 mM KCl, 3 mM MgCl2, 2 mM DTT,Rabbit RL (Hsp90, 2 µM; Hsc70, 2~4.2 µM; Hsp40, 0.15 µM) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.2 |
| Refolding Temperature | 30.0 |
| Protein Concentration | 0.26 uM |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 2 mM |
| Refolding Protocol | Firefly luciferase (Sigma) was thermally inactivated for 5 min at 45 or 50 °C, or for 10 min at 42 °C at a final concentration of 0.26 µM in 30 mM MOPS-KOH, pH 7.2, 2 mM dithiothreitol (DTT) solution containing either Hsp90 or bovine serum albumin (BSA) as indicated. For refolding assays, thermally inactivated luciferase was diluted 10-fold into refolding buffer (10 mM MOPS-KOH, pH 7.2, 50 mM KCl, 3 mM MgCl2, 2 mM DTT) containing the indicated components. Rabbit RL was obtained from Promega. The approximate concentrations of chaperones in RL are reported to be: Hsp90, 2 µM; Hsc70, 2~4.2 µM; Hsp40, 0.15 µM (30, 31). Aliquots (2 µl) were withdrawn from the reaction mixtures after incubation for 30 min, 1, or 1.5 h as indicated at 30 °C and mixed with a Promega luciferase assay system (40 µl). Luciferase activities were immediately measured by a Bio-Orbit 1253 luminometer and expressed as the percentage of the activity before thermal inactivation. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | Rabbit RL (Hsp90,Hsc70,Hsp40) |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |