Refolding Record:
| Protein | |
|---|---|
| Protein Name | Gamma-interferon-induced monokine |
| Abbreviated Name | MuMIG |
| SCOP Family | interleukin 8-like chemokines |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | P18340 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 105 |
| Molecular Weight | 12194.3 |
| Pi | 10.6 |
| Molecular Weight | 12194.3 |
| Disulphides | 2 |
| Full Sequence |
TLVIRNARCSCISTSRGTIHYKSLKDLKQFAPSPNCNKTEIIATLKNGDQTCLDPDSANVKKLMKEWEKKINQKKKQKRGKKHQKNMKNRKPKTPQSRRRSRKTT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lu H, YU M, Sun Y, Mao W, Wang Q, Wu M, Han W (2007) Protein Expression and Purification, 55, 132-138 |
| Project Aim | Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pET28a-m |
| Expression Protocol | Eight hundred milliliters of LB medium, supplemented with kanamycin (100 μg/ml), was inoculated with an overnight starter culture (OD600 of <0.1). Cultures were grown at 37 °C with 250 rpm shaking to OD600 of 0.8–1.0 and then induced with 1 mM IPTG (final concentration) for 3 h. Cells were harvested by centrifugation at 10,000g for 15 min. Cell pellet was re-suspended in the lysis buffer (1 × PBS, pH 7.4; 1 mM EDTA; 0.1 mM PMSF) at a final concentration of 50 mg/ml and sonicated (SANYO soniprep 150, 15 bar; 30 s working and 30 s resting on ice for a cycle, 18 cycles). After centrifugation at 12,000g for 15 min at 4 °C, the undissolved inclusion bodies were collected and washed with the pellet wash buffer (1 × PBS, pH 7.4; 0.5% Triton X-100; 10 mM EDTA; 50 mM NaCl) at 12,000g for 5 min at 4 °C, 5× |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8-1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 1 × PBS, pH 7.4; 0.5% Triton X-100; 10 mM EDTA; 50 mM NaCL |
| Solubilization Buffer | 20 mM Na2HPO4, 1 mM EDTA, 50 mM NaCl, pH 9.0, 8 M Urea (9 ml Urea/g pellet) |
| Refolding Buffer | 20 mM NaPO4, 1 mM EDTA, pH 6.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a,n/a |
| Refolding Protocol | Cells were harvested by centrifugation at 10,000g for 15 min. Cell pellet was re-suspended in the lysis buffer (1 × PBS, pH 7.4; 1 mM EDTA; 0.1 mM PMSF) at a final concentration of 50 mg/ml and sonicated (SANYO soniprep 150, 15 bar; 30 s working and 30 s resting on ice for a cycle, 18 cycles). After centrifugation at 12,000g for 15 min at 4 °C, the undissolved inclusion bodies were collected and washed with the pellet wash buffer (1 × PBS, pH 7.4; 0.5% Triton X-100; 10 mM EDTA; 50 mM NaCl) at 12,000g for 5 min at 4 °C, 5×. The inclusion bodies were then solubilized in Buffer U (20 mM Na2HPO4, 1 mM EDTA, 50 mM NaCl, pH 9.0) containing 8 M urea at 9 ml Urea/g pellet, shaking slightly at 37 °C for 1 h. The insoluble pellets were removed by centrifugation at 15,000g for 20 min at 4 °C, and the dissolved inclusion bodies were ready for further purification. Refolding and purification of rMuMIG The refolding was proceeded by drop-wise dilution into defined protein folding buffer. The protein solution was dropped by pumping into 10-fold volume refolding Buffer (20 mM NaPO4, 1 mM EDTA, pH 6.0), under vigorous (magnetic stirrer) agitation. pH of the solution was maintained at 6.0. Then an equal volume dilution Buffer (20 mM NaPO4, 1 mM EDTA, pH 8.5) was added into the folding buffer. The refolded protein solution was set at 4 °C for about 24 h. The refolded protein solution was centrifuge at 18,000g for 30 min. The collected supernatant was loaded on an S-Sepharose column with a volume of approximately 20 ml. The column was pre-equilibrated with Buffer A (20 mM NaPO4, 1 mM EDTA, pH 7.2). Sample was loaded at speed of 0.5 ml/min and the column was then washed with 2 column volumes of Buffer A. The column bound proteins were eluted using a programmed gradient of Buffer B (20 mM NaPO4, 1 mM EDTA, 1 M NaCl, pH 7.2) at a speed of 3 ml/min. Fractions containing the rMuMIG protein were identified by the Bradford method . |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 5.2% |
| Purity | n/a |
| Notes | note refolding PH shift from 6 to 8.5 |