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 | 140 |
| Molecular Weight | 16177.5 |
| Pi | 9.52198 |
| Molecular Weight | 16177.5 |
| Disulphides | 0 |
| Full Sequence |
QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWK
EESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN
YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Xindu Geng, Quan Bai, Yangjun Zhang, Xiang Li, Dan Wu (2004) J Biotechnology, 113, 137-149 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5? |
| Expression Temp | 37.0 |
| Expression Time | 12h |
| Expression Vector | pBV 220 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: hydrophobic interaction chromatography |
| Wash Buffer | 2M urea + 50 mM PBS, pH7.0 |
| Solubilization Buffer | 7.0 mol L?1 GuHCl + 50 mM PBS, pH7.0 |
| Refolding Buffer | 50mM PBS |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 4h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The method for producing the inclusion body of rhIFN-gamma (pBV 220/DH5?) expressed by E. coli was taken from the Doctorial thesis by Shen (2001). The bacteria was produced with a 50 L fermenter (B.Brauwn Co, Germany). The yield of the rhIFN- 14.0 g dry cell L?1 with fed-batch fermentation. The bacteria was put into buffer A and crashed by ultrasonic processor in an ice-water bath and then centrifuged at 18,000 rpm for 15 min. The isolated inclusion bodywas washed once for each of buffer B, buffer C and buffer D, respectively. After that, the clean inclusion body was dissolved in 7.0 mol L?1 GuHCl solution. After incubation at 4 ?C for 24 h with full agitation, the extract of the rhIFN-gamma was obtained by centrifuging at 20,000 rpm. The unit of simultaneous renaturation and purification of proteins (USRPP) and chromatographic columns of HPHIC were initially equilibrated with solution A, at least for 15 min at each selected flow-rate before injecting a sample solution and then a linear gradient elution of 0?00% solution B at different times was performed at a selected flow rate and detected at 280 nm. The selection of flow rate depends on the size of column, or the USRPP and detected at 280 nm. The eluted fractions of the aim proteins were collected for the measurements of the recoveries of bioactivity and mass of the rhIFN-gamma. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | 95% |
| Notes | |