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 | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 146 |
| Molecular Weight | 17145.6 |
| Pi | 9.54 |
| Molecular Weight | 17145.6 |
| Disulphides | 0 |
| Full Sequence |
CYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ
|
| Notes | No details of construct sequence given. Sequence provided is the sequence for mature IFN-gamma |
| Expression | |
|---|---|
| Report | Arora, D., Khanna, N. (1996) J Biotechnology, 52, 127-133 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 42.0 |
| Expression Time | 4h |
| Expression Vector | c1857 |
| Expression Protocol | 20 ml of M9+CAA containig 150 microg/ml ampicillin was inoculated with overnight culture grown at 29 |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M GuHCL per 0.1 M TrisHCL, pH 8.0 per 0.2 mM EDTA, pH 8.0 |
| Refolding Buffer | 0.1 M Tris, pH 8.0, 0.2 mM EDTA with or without arginine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | 36-48 |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were disolved in 20 ml solubilization buffer and stired in small beaker for two hours at room temperature. Solution then centrifuged(17,000rpm, 30min, 4degC.)The supernatant diluted to 35 ml for final protein concentration of 10 mg/ml. Renaturation was preformed by dilution in refolding buffer. The renatured protein in the presence of 0.5 M l-arginine was dialysed against 20mM TrisHCl, pH 8.0, containing 100mM urea at 10degC until conductivity was between 3-3.7 mMhos. Solution then centrifuged(10,000rpm, 10 min) and supernatant collected. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |