Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interferon gamma receptor |
| Abbreviated Name | IFNgamma-R |
| SCOP Family | Fibronectin type III |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P15260 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | aa.15-246 of premature protein |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 242 |
| Molecular Weight | 27513.0 |
| Pi | 5.63874 |
| Molecular Weight | 27513.0 |
| Disulphides | 4 |
| Full Sequence |
MRG RAEMG TADLGPSSVP TPTNVTIESY NMNPIVYWEY QIMPQVPVFT VEVKNYGVKN SEWIDACINI SHHYCNISDH VGDPSNSLWV RVKARVGQKE SAYAKSEEFA VCRDGKIGPP KLDIRKEEKQ IMIDIFHPSV FVNGDEQEVD YDPETTCYIR VYNVYVRMNG SEIQYKILTQ KEDDCDEIQC QLAIPVSSLN SQYCVSAEGV LHVWGVTTEK SKEVCITIFN SSIKGS RSHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fountoulakis M, Juranville J-F, Stuber D, Weibel EK, Garotta G (1990) J Biol Chem, 265, 13268-13275 |
| Project Aim | Functional Studies |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pDS56 |
| Expression Protocol | Cells werer grown in L-broth medium containing 100mg/mL ampicillin and 25mg/L kanamycin. When A580 reached 0.8, expression was induced by the addition of 0.5mM IPTG. Cells were grown for a further 3h then harvested by centrifugation and stored at -70degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 580 = 0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 0.1M phosphate buffer pH 8.0, 7M guanidine |
| Refolding Buffer | 0.1M Tris pH 9.0, 10% glycerol, 10mM benzamidine HCl, 1mM PMSF, 1ooU/ml aprotinin |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | 100g of cells were thawed at 4degC overnight, then suspended in 1L of solubilization buffer with the use of a homogenizer for 10min. The cell suspension was sonicated for 5min, kept in ice for one hour and centrifuged (12000g, 60min). The supernatant was loaded onto a Ni-NTA column equilibrated with solubilization buffer at 50ml/hr. After loading, the column was washed with 10 column volumes(CV) of solubilization buffer, then with 10CV of 0.1M TrisHCl pH 8.0, 7M urea. The proteins were eluted with a step gradient of decreasing pH from 8.0 to 4.0 (0.1M Tris-maleate or glycine-HCl containing 7M urea). Selected fractions were pooled, concentrated from 1000ml to 20ml, and then loaded onto a Sephadex G-100 column equilibrated with 50mM TrisHCl pH 7.5, 7M urea. The column was eluted wtih the same buffer at 30ml/hr and selected fractions were pooled. The collected fractions were then dialyzed against refolding buffer. The dialysis was then continued against phosphate-buffered saline pH 7.4. The refolded protein was then loaded onto a Polybuffer Exchanger column, followed by a Sephadex G-100 column. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | 25-30% |
| Purity | 80-90% |
| Notes | |