Refolding Record:
| Protein | |
|---|---|
| Protein Name | Aequorin |
| Abbreviated Name | Aequorin |
| SCOP Family | Calmodulin-like |
| Structure Notes | |
| Organism | Aqueorea victoria |
| UniProt Accession | P07164 |
| 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 | 196 |
| Molecular Weight | 22641.4 |
| Pi | 5.30357 |
| Molecular Weight | 22641.4 |
| Disulphides | 0 |
| Full Sequence |
MHHHHHH VKLTPDFDNPKWI GRHKHMFNFL DVNHNGRISL DEMVYKASDI VINNLGATPE QAKRHKDAVE AFFGGAGMKY GVETEWPEYI EGWKRLASEE LKRYSKNQIT LIRLWGDALF DIIDKDQNGA ISLDEWKAYT KSDGIIQSSE DCEETFRVCD IDESGQLDVD EMTRQHLGFW YTMDPACEKL YGGAVP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Glynou K, Ioannou PC, Christopoulos TK (2003) Protein Expression and Purification, 27, 384-390 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pKK223-3 |
| Expression Protocol | 100ml of LB broth containing 50 ìg/ml ampicillin was inoculated with 1 ml from an overnight culture. Cells were grown at 37degC to an absorbance value of 0.5 (600 nm). IPTG was then added to a final concentration of 0.1 mM. After 4 h of induction at 37degC, the medium was cooled down and the cells were harvested by centrifugation at 4000g for 20 min at 4degC. The supernatant was discarded and the pellet was stored at −20degC overnight. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 10mM Tris-HCl, 6M urea, 2.5mM imidazole, 0.5M NaCl, 10mM beta mercaptoethanol, 20% glycerol pH 7.8 |
| Refolding Buffer | 10mM Tris-HCl, 6M urea, 2.5mM imidazole, 0.5M NaCl, 10mM beta mercaptoethanol, 20% glycerol |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.8 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 1h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 10mM |
| Refolding Protocol | Refolding was carried out on a Ni-NTA column. The urea was removed slowly over the course of an hour using a 4 ml (10 bed volumes) gradient of 6M urea to 0M urea. |
| Refolding Assay | Luminescence |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | 13.4mg/1L culture |
| Purity | |
| Notes | |