Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-4 |
| Abbreviated Name | IL-4 |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P05112 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 130 |
| Molecular Weight | 14963.2 |
| Pi | 9.25 |
| Molecular Weight | 14963.2 |
| Disulphides | 3 |
| Full Sequence |
HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | van Kimmenade A, Bond MW, Schumacher JH, Laquoi C, Kastelein RA. (1988) Eur J Biochem., 173, 109-114 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | AB1899 |
| Expression Temp | 37.0 |
| Expression Time | 10 |
| Expression Vector | n/a |
| Expression Protocol | E. coli AB1899 cells carrying the TrpC11-hIL4 plasmid were grown in L-broth (1 : 50 dilution of an overnight culture) to a total absorbance at 560 nm of 2; 1 ml of this culture was centrifuged for 5 min and the cells were resuspended in 500 ul phosphate-buffered saline. Cells were then lysed by sonication (30 - 50% pulses, 40 W) using a Branson cell disrupter 200. After centrifugation, the pellet was resuspended in 100 ul phosphate-buffered saline containing 1 % SDS and incubated for 10 min at 37°C. The proteins solubilized with 1% SDS were separated from the insoluble fraction by centrifugation for 10 min at 4°C. Centrifugations were performed at 15000 x g. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 5 M guanidine . HC1 and 2 mM reduced and 0.2 mM oxidized glutathione |
| Refolding Buffer | 50 mM Tris/HCl pH 8.0,50 mM NaCI, 1 mM EDTA, 2 mM reduced and 0.2 mM oxidized glutathione |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 1.5-4 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/0.2 mM |
| Refolding Protocol | One liter of E. coEi AB1899 cells harboring the TrpC11- hIL4 plasmid were grown (1 : 50 dilution of an overnight culture) in a 2-1 conical flask at 37°C in L-broth for 10 h. Cells were harvested by centrifugation at 4500 x g for 15 min at 4°C and stored at -20°C overnight. To lyse the cells, the pellets were resuspended in 30 ml 50 mM Tris/HCl pH 8.0,50 mM NaCI, 1 mM EDTA, 0.1 mM henylmethylsulphonyl fluoride (buffer A), and sonicated (50 50% pulses, 70 W). The cell lysates were centrifuged at 25000 xg for 15 min at 4°C. The resulting pellet containing the insoluble human IL-4 protein was resuspended in buffer A supplemented with 5 M guanidine . HC1 and 2 mM reduced and 0.2 mM oxidized glutathione. For every 1 g wet pellet, 9 ml buffer was added to a maximum protein concentration of 2.5 mg/ml. After 1 hat room temperature, this solution was added slowly to 9 vol. buffer A (without phenylmethylsulfonyl fluoride) supplemented with 2 mM reduced and 0.2 mM oxidized glutathione, and incubated at room temperature for 1.5-4 h.The lysate was centrifuged at 2500 x g for 15 min at 4°C to remove the precipitate formed during the dilution step. The clear supernatant was dialyzed against phosphate-buffered saline pH 7.4 with three changes of buffer (total dilution 1000- fold) at 4°C to remove the refolding reagents. After removal by centrifugation of the precipitate formed during dialysis, the protein concentration of the dialyzate was increased to 8 mg/ml using an Amicon concentrator with an YM5 filter. Again precipitates formed wich were removed by centrifugation. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 46% |
| Purity | n/a |
| Notes | n/a |