Refolding Record:
| Protein | |
|---|---|
| Protein Name | Steroleosin |
| Abbreviated Name | sop2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Sesamum indicum (Oriental sesame) (Gingelly) |
| UniProt Accession | Q93W57 |
| 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 | 348 |
| Molecular Weight | 39567.6 |
| Pi | 5.71 |
| Molecular Weight | 39567.6 |
| Disulphides | Unknown |
| Full Sequence |
MDLIHTFLNLIAPPFTFFFLLFFLPPFQIFKFFLSILGTLFSEDVAGKVVVITGASSGIGESLAYEYAKRGACLVLAARRERSLQEVAERARDLGSPDVVVVRADVSKAEDCRKVVDQTMNRFGRLDHLVNNAGIMSVSMLEEVEDITGYRETMDINFWGYVYMTRFAAPYLRNSRGRIVVLSSSSSWMPTPRMSFYNASKAAISQFFETLRVEFGPDIGITLVTPGFIESELTQGKFYNAGERVIDQDMRDVQVSTTPILRVESAARSIVRSAIRGERYVTEPAWFRVTYWWKLFCPEVMEWVFRLMYLASPGEPEKETFGKKVLDYTGVKSLLYPETVQVPEPKND
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lin LJ, Tai SS, Peng CC, Tzen JT (2002) Plant Physiol, 128, 1200-1211 |
| 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 | NovaBlue |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pQE30b |
| Expression Protocol | Overexpression of the Sesame Sop2 Clone in Escherichia coli The cDNA fragment encoding the sterol-binding dehydrogenase/reductase domain of sesame Sop2 was constructed in the fusion expression vector, pQE30b(+) (QIAGEN, Valencia, CA), using a BamHI and a HindIII site in the polylinker of the vector. The recombinant plasmid was used to transform E. coli strain NovaBlue. Overexpression was induced by 0.1 mm isopropyl β-d-thiogalactoside in a bacteriophage T7 RNA polymerase/promoter system. Three hours after induction, the E. coli cells were harvested and crashed by sonication in the extraction/washing buffer containing 300 mm NaCl and 50 mm NaH2PO4, pH 7.0. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 300 mm NaCl and 50 mm NaH2PO4, pH 7.0. |
| Solubilization Buffer | 7 M urea |
| Refolding Buffer | 10 mm sodium phosphate buffer, pH 7.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 4 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The sonicate was clarified by centrifugation at 14,000 rpm at 4°C for 15 min. The pellet was resuspended in 7 m urea and was dialyzed against 10 mm sodium phosphate buffer, pH 7.5, for 4 h at 4°C. After dialysis, the sample was centrifuged at 14,000 rpm at 4°C for 5 min and the supernatant was incubated with TALON resin (CLONTECH, Palo Alto, CA) for 20 min at room temperature. The resin was then spun down at 700g and was washed with the extraction/washing buffer by gentle end-over-end mixing at 4°C for 10 min. The His-tagged protein was eluted with the imidazole elution buffer containing 150 mm imidazole in extraction/washing buffer. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |