Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interferon gamma |
| Abbreviated Name | IFN gamma |
| SCOP Family | Interferons/interleukin-10 (IL-10) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01579 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 140 |
| Molecular Weight | 16177.5 |
| Pi | 9.52 |
| Molecular Weight | 16177.5 |
| Disulphides | Unknown |
| Full Sequence |
QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Guan YX, Fei ZZ, Luo M, Jin T, Yao SJ. (2006) J Chromatogr A., 1107, 192-197 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | n/a |
| Expression Protocol | The rhIFN-γ was expressed in an insoluble form as inclusion bodies, as previously reported [20]. The cell pellets were collected by centrifugation, resuspended in 100 ml of 50 mM Tris buffer pH 8.0 (1 mM EDTA) and lysed by ultrasonication on ice bath. Cellular lysates were centrifuged at 8000 × g for 15 min. Then inclusion bodies were washed with 15 ml of 50 mM Tris buffer pH 8.0 (0.5% Triton X-100) and incubated at 37 °C for 2 h. Finally the pellets were solubilized in 50 ml denaturation buffer pH 7.7 (30 mM sodium phosphate, 8 M urea, 10 mM DTT, 1 mM EDTA and 150 mM NaCl) at 37 °C for 12 h. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/ chaperone-assisted refolding |
| Wash Buffer | 50 mM Tris buffer pH 8.0 (0.5% Triton X-100 |
| Solubilization Buffer | buffer pH 7.7 (30 mM sodium phosphate, 8 M urea, 10 mM DTT, 1 mM EDTA and 150 mM NaCl) |
| Refolding Buffer | 150 mM NaCl and 1 M urea, 5 ml immobilized sht GroEL191-345 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 2 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Chromatographic refolding was conducted on a 5 ml immobilized sht GroEL191-345 column at room temperature. Firstly the column was equilibrated with urea, then denatured rhIFN-γ sample of 17.8 mg/ml was applied directly to the column at a flow rate of 1 ml/min and eluted with the refolding buffer containing 150 mM NaCl and 1 M urea. The fractions were pooled for dialysis after refolding. In the experiment, the rhIFN-γ refolding with an immobilized chaperone fragment column chromatography was performed on ÄKTA Explorer 100. Each experiment was carried out in triplicate, the mean values were adopted. The effects of NaCl concentration in the equilibrium buffer (150, 300 and 500 mM), pre-equilibrium mode (urea non-gradient of buffer A containing 500 mM NaCl and 1 M urea, and a linear urea gradient of buffer A containing 500 mM NaCl and 1 M urea and buffer B containing 500 mM NaCl and 8 M urea), elution flow rate (0.025, 0.05, 0.1 and 0.2 ml/min) and loading amount (50, 100, 200 and 500 μl) on the refolding were investigated in detail. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | sht GroEL191-345 |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |