Refolding Record:
| Protein | |
|---|---|
| Protein Name | apoptotic protease activating factor |
| Abbreviated Name | Apaf-1 |
| SCOP Family | Caspase recruitment domain, CARD |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O14727 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 450 |
| Molecular Weight | 51126.0 |
| Pi | 6.44 |
| Molecular Weight | 51126.0 |
| Disulphides | Unknown |
| Full Sequence |
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWIMDAKARNCLLQHREALEKDIKTSYIMDHMISDGFLTISEEEKPTQQQRAAMLIKMILKKDNDSYVSFYNALLHEGYKDLAALLHDGIPVVSSSSGKDSVSGITSYVRTVLCEGGVPQRPVVFVTRKKLVNAIQQKLSKLKGEPGWVTIHGMAGCGKSVLAAEAVRDHSLLEGCFPGGVHWVSVGKQDKSGLLMKLQNLCTRLDQDESFSQRLPLNIEEAKDRLRILMLRKHPRSLLILDDVWDSWVLKAFDSQCQILLTTRDKSVTDSVMGPKYVVPVESSLGKEKGLEILSLFVNMKKADLPEQAHSIIKECKGSPLVVSLIGALLRDFPNRWEYYLKQLQNKQFKRIRKSSSYDYEALDEAMSISVEMLREDIKDYYTDLSILQKDVKVPTKVLCILWDMETEEVEDILQEFV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Raoa, PN., Yadaiaha, M., Royb, KR., Potua, H & Bhuyan, AK (2007) Protein Expression and Purification, 56, 220-228 |
| 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 | DH5alpha |
| Expression Temp | 37.0 |
| Expression Time | 5 |
| Expression Vector | pRSETA |
| Expression Protocol | Typically, 20 ml of an overnight grown culture was added to a 2-L of media, and incubated with vigorous shaking at a temperature of 37 °C. At OD600 = 0.5 of the culture, protein expression was induced with 1mM IPTG. The growth was continued for 5h after induction. Cells were harvested by centrifugation at 4000g for 10 min at 4 °C, washed with TE buffer (10 mM Tris–HCl, 1 mM EDTA pH 8.0), and frozen stored at −80 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM Tris, 100 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, pH 8.0 |
| Solubilization Buffer | 6.0 M GdnHCl, 20 mM Tris–HCl, 500 mM NaCl, pH 7.5 1mM PMSF 5 mM β-mercaptoethanol, pH 8.0 |
| Refolding Buffer | 0.4 M arginine, 0.5 M urea, and 1 mM β-mercaptoethanol, pH 7.5 20 mM Tris–HCl, 500 mM NaCl, pH 7.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | nclusion bodies were solubilized in Buffer C with 1 mM PMSF and 5 mM β-mercaptoethanol, pH 8.0, incubated for 2 h, and refolded by 20-fold dilution into Buffer C containing no GdnHCl but 0.4 M l-arginine, 0.5 M urea, and 1 mM β-mercaptoethanol, pH 7.5. The solution was kept in cold for 24 h, and then dialyzed extensively against the same refolding buffer (pH 8.0) that excluded l-arginine and β-mercaptoethanol. After discarding the precipitate by centrifugation, the solution was loaded onto a Ni-NTA-His bind column (Novagen) equilibrated with the dialysis buffer, and washed with the same buffer. The protein, eluted by passing 20 mM Tris, 150 mM NaCl, and 300 mM imidazole, pH 8, was dialyzed or centrifuged in Amicon 5 kDa cutoff filter devices to remove imidazole. Although nothing wrong in itself, this procedure for imidazole removal was time consuming under our laboratory conditions. We, therefore, used 20 mM Tris, 150 mM NaCl, 50 mM EDTA, pH 8 for protein elution. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.4M |
| Refolding Yield | 1.2mg/l |
| Purity | n/a |
| Notes | n/a |