Refolding Record:
| Protein | |
|---|---|
| Protein Name | Farnesoid X-activated receptor |
| Abbreviated Name | FXR |
| SCOP Family | Nuclear receptor ligand-binding domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q96RI1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | ligand binding domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 231 |
| Molecular Weight | 26892.8 |
| Pi | 5.63 |
| Molecular Weight | 26892.8 |
| Disulphides | Unknown |
| Full Sequence |
DLRQVTSTTKSCREKTELTPDQQTLLHFIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATNHVQVLVEFTKKLPGFQTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPSGHSDLLEERIRNSGISDEYITPMFSFYKSIGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKIHQPENPQHFACLLGRLTELRTFNHHHAEMLMSWRVND
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Schoner BE, Bramlett KS, Guo H, Burris TP. (2005) Molecular genetics and metabolism, 85, 318-322 |
| Project Aim | Protein refolding |
| Fusion | N-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 | 3-5 h |
| Expression Vector | pET-based plasmid |
| Expression Protocol | Overnight cultures grown at 37 °C in TY broth (supplemented with 100 μg/ml ampicillin) were diluted 1:50 into fresh broth and growth was continued at 37 °C until an OD600 of 0.8 was reached. IPTG (Invitrogen Life Technologies, Carlsbad, CA) was added to a final concentration of 0.5 mM and growth was continued for an additional 3–5 h at which time the cells were harvested by centrifugation, and the cell pellets were stored at −20 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | High Pressure |
| Wash Buffer | n/a |
| Solubilization Buffer | n/a |
| Refolding Buffer | not specified |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 0.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 16 h |
| Redox Agent | Not specified |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | For the high-pressure refolding, the purified inclusion bodies were packaged into a sealed plastic bag. After placing the bags inside the oil-containing pressure cell, the samples were slowly pressurized to 2.5 kbar, and kept at that pressure for 16 h at room temperature. Depressurization was at a rate of 200 bars/15 min |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |