Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-4 |
| Abbreviated Name | IL-4 |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | P07750 |
| 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 | 124 |
| Molecular Weight | 14033.3 |
| Pi | 8.56 |
| Molecular Weight | 14033.3 |
| Disulphides | Unknown |
| Full Sequence |
MTRS HIHGCDKNHL REIIGILNEV TGEGTPCTEM DVPNVLTATK NTTESELVCR ASKVLRIFYL KHGKTPCLKK NSSVLMELQR LFRAFRCLDS SISCTMNESK STSLKDFLES LKSIMQMDYS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Levine AD, Rangwala SH, Horn NA, Peel MA, Matthews BK, Leimgruber RM, Manning JA, Bishop BF, Olins PO. (1995) J Biol Chem, 270, 7445-7452 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM101 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pMON 5743 |
| Expression Protocol | Cells were grown at 37degC in M9 medium. When OD550 reached 0.5, expression was induced with 50microg/ml nalidixic acid and growth was continued for a further 4h. Cells were harvested by centrifugation |
| Method of Induction | Nalidixic Acid |
| Cell Density at Induction | OD 550 = 0.5 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris-HCl |
| Solubilization Buffer | 50mM CHES, 4M guanidinium chloride, 0.5M NaCl, 50mM DTT, pH 9.5 |
| Refolding Buffer | 50mM CHES, 0.5M NaCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 9.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 34h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 0.02M |
| Refolding Protocol | All steps were performed at room temperature. The washed inclusion bodies were dissolved (15-20 g pellet/liter) in buffer B (4 M guanidine, 0.5 M NaCl, 0.05 M DTT, and 0.05 M CHES, pH 9.5, for 4-6 h. This solution was loaded into Spectra/Por dialysis membrane tubing (molecular weight cutoff, 3500) previously washed in Milli-Q HO and dialyzed against 15 volumes of 4 M guanidine, 0.5 M NaCl, 0.02 M cysteine, 0.05 M CHES, pH 9.5 (buffer C) to begin slow disulfide formation. After 12-15 h of dialysis in buffer C, the dialysis buffer was changed to 4 M guanidine, 0.5 M NaCl, 0.05 M CHES, pH 9.5 (buffer D) to initiate fast disulfide formation. Buffer D was changed after 8-12 h. Oxidation was complete within 24 h after dialysis against buffer D. Oxidized IL-4 was refolded, partially purified, and prepared for ion-exchange chromatography by two negative precipitation steps. First, the guanidine was removed by dialysis against 15 volumes of buffer D with two buffer changes (8-12 h apart). The salt was then removed by dialysis against 15 volumes of 0.05 M CHES, pH 9.5 (buffer E). A heavy cream-colored precipitate formed in the dialysis bag. The refold solution was clarified by centrifugation in a Sorvall RC5C centrifuge with a GS-3 rotor at 10,000 rpm (16,900 g) for 30 min in preparation for the second precipitation step. The supernatant was loaded into Spectra/Por dialysis membrane tubing (molecular weight cutoff, 3500) previously washed in Milli-Q HO and dialyzed against 15 volumes buffer F (0.05 M acetic acid adjusted to pH 5.0 with NaOH) at 4-10 °C with two exchanges (8-12 h apart). A white precipitate formed in the dialysis bag. The acidified refold solution was clarified by centrifugation in a Sorvall RC5C centrifuge with a GS-3 rotor at 10,000 rpm (16,900 g) for 30 min. The supernatant was filtered through a 0.22-µm cellulose acetate membrane and stored at 4-10 °C. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 10-15% |
| Purity | a few c |
| Notes | |