Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interferon regulatory factor 4 |
| Abbreviated Name | IRF-4 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q15306 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 451 |
| Molecular Weight | 51772.5 |
| Pi | 6.3923 |
| Molecular Weight | 51772.5 |
| Disulphides | 0 |
| Full Sequence |
MNLEGGGRGG EFGMSAVSCG NGKLRQWLID QIDSGKYPGL VWENEEKSIF RIPWKHAGKQ DYNREEDAAL FKAWALFKGK FREGIDKPDP PTWKTRLRCA LNKSNDFEEL VERSQLDISD PYKVYRIVPE GAKKGAKQLT LEDPQMSMSH PYTMTTPYPS LPAQQVHNYM MPPLDRSWRD YVPDQPHPEI PYQCPMTFGP RGHHWQGPAC ENGCQVTGTF YACAPPESQA PGVPTEPSIR SAEALAFSDC RLHICLYYRE ILVKELTTSS PEGCRISHGH TYDASNLDQV LFPYPEDNGQ RKNIEKLLSH LERGVVLWMA PDGLYAKRLC QSRIYWDGPL ALCNDRPNKL ERDQTCKLFD TQQFLSELQA FAHHGRSLPR FQVTLCFGEE FPDPQRQRKL ITAHVEPLLA RQLYYFAQQN SGHFLRGYDL PEHISNPEDY HRSIRHSSIQ E
|
| Notes | Sequence does not include N-terminal his tag |
| Expression | |
|---|---|
| Report | Moellering MJ, Yoshinaga SK, Hui A, Delaney JM, Hara S, Narhi LO, Westcott KR (1999) Protein Expression and Purification, 16, 160-170 |
| Project Aim | Structure-Function |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | K12 |
| Expression Temp | 37.0 |
| Expression Time | 12h |
| Expression Vector | pAMG21 |
| Expression Protocol | Cells were grown to mid-log phase at 37degC, expression was induced by the addition of N-(beta-ketocaproyl)-DL-homoserine lactone (0.5ng/ml). After 12h, the cells were harvested by centrifugation. |
| Method of Induction | N-(beta-ketocaproyl)-DL-homoserine lactone |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Diafiltration |
| Wash Buffer | n/a |
| Solubilization Buffer | 25mM TrisHCl pH 8.5, 8M GdnHCl, 5mM DTT, 2mM EDTA |
| Refolding Buffer | 25mM Hepes pH 8.5, 60mM KCl, 400mM NaCl, 5mM imidazole |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were suspended in 10 volumes of 25mM TrisHCl pH 8.5, 5mM DTT, 5mM EDTA. The cell suspension was homogenized and the cells were passed three times through a microfluidizer at 15000psi. The inclusion bodies were dissolved in solubilization buffer, the suspension was homogenized and allowed to stir for 20min prior to contrifugation (28000g, 45min). The supernatant was diafiltered into 25mM TrisHCl pH 8.5, 5mM DTT, 2mM EDTA, 6M urea and then centrifuged. The supernatant was then diafiltrated into refolding buffer. The protein was centrifuged again (28000g, 45min) and then purified using Ni-IMAC, hydrophobic interaction and gel filtration chromatography. |
| Refolding Assay | Far-UV Circular Dichroism |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |