Refolding Record:
| Protein | |
|---|---|
| Protein Name | Leukotriene B4 receptor 1 |
| Abbreviated Name | BLT1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q15722 |
| 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 | 370 |
| Molecular Weight | 39552.6 |
| Pi | 10.8 |
| Molecular Weight | 39552.6 |
| Disulphides | 0 |
| Full Sequence |
MNTTSSAAPPSLGVEFISLLAIILLSVALAVGLPGNSFVVWSILKRMQKRSVTALMVLNLALADLAVLLTAPFFLHFLAQGTWSFGLAGCRLCHYVCGVSMYASVLLITAMSLDRSLAVARPFVSQKLRTKAMARRVLAGIWVLSFLLATPVLAYRTVVPWKTNMSLCFPRYPSEGHRAFHLIFEAVTGFLLPFLAVVASYSDIGRRLQARRFRRSRRTGRLVVLIILTFAAFWLPYHVVNLAEAGRALAGQAAGLGLVGKRLSLARNVLIALAFLSSSVNPVLYACAGGGLLRSAGVGFVAKLLEGTGSEASSTRRGGSLGQTARSGPAALEPGPSESLTASSPLKLNELNSSSVDKLAAALEHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Baneres, J.L., Martin, A., Hullot, P., Girard, J.P., Rossi, J.C., Parello, J. (2003) J Mol Biol, 329, 801-814 |
| Project Aim | Structure-Function |
| Fusion | C-terminal hexahistidine tag |
| 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 | 4h |
| Expression Vector | pET21b |
| Expression Protocol | Cells were grown in 2YT medium containing 100mg/L of ampicillin. Induced cells were harvested by cen |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M urea, 25mM TrisHCl, pH 7.8, containing 500mM NaCl, 10microg/ml leupeptin, 10microgram/ml; aproptinin and 0.5mM PMSF |
| Refolding Buffer | 6-0 M urea linear gradient, 12.5mM Na-borate, 10% (v/v) glycerol, 50mM KCL, 2 mM LDAO, 2.5mM reduced glutathione, 0.25mM oxidized glutathione, pH 8.0 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 20.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Not specified |
| Redox Agent Concentration | n/a |
| Refolding Protocol | His tagged BLT1 was imobilized on a Ni-NTA agarose matrix and and refolded using a 6-0 M linear urea gradient in refolding buffer. Dissociation was achieved with refolding buffer and 100mM imidazol and eluted protein was centrifuged(150,000g, 60min, 20degC) to remove aggregated material. C terminal His Tag was removed by digestion with thrombin at 20degC and uncleaved protein was removed by running through Ni-NTA column. |
| Refolding Assay | Fluorescence,Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% v/v |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |