Refolding Record:
| Protein | |
|---|---|
| Protein Name | General odorant-binding protein 2 |
| Abbreviated Name | GOBP2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm |
| UniProt Accession | P31419 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 141 |
| Molecular Weight | 16285.7 |
| Pi | 5.21 |
| Molecular Weight | 16285.7 |
| Disulphides | 3 |
| Full Sequence |
TAEVMSHVTAHFGKALEECREESGLPVEVMDEFKHFWREDFEVVHRELGCAIICMSNKFELLQDDTRIHHVKMHDYIKSFPNGQVLSEKMVQLIHNCEKQYDDIADDCDRVVKVAACFKKDAKKEGIAPEVAMIEAVIEKY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Feng L, Prestwich GD. (1997) Insect Biochem Mol Biol., 27, 405-12 |
| Project Aim | Purification & characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XLBlue |
| Expression Temp | 37.0 |
| Expression Time | 5-6 h |
| Expression Vector | n/a |
| Expression Protocol | For large-scale protein production, 1 1 of LB-ampicillin was inoculated with a 5 ml overnight culture and induced by 1 mm IPTG when OD6oo was approximately 0.4~).6. After 5-6 h, cells were isolated by centrifugation |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4-0.6 = 600 |
| Cell Disruption Method | Sonication/French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.2% Triton X-100 in 50 mm Tris buffer (pH 6.8) |
| Solubilization Buffer | 5 ml of 6 N guanidinium hydrochloride |
| Refolding Buffer | 5 mM cysteine in 100 mM Tris-HCI, pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | Protein refolding Insoluble protein from a 1 1 culture was washed with 0.2% Triton X-100 in 50 mm Tris buffer (pH 6.8) and then dissolved in 5 ml of 6 N guanidinium hydrochloride. A redox protocol (De Bernardez-Clark and Georgiou, 1991) as modified for refolding PBPs (Prestwich, 1993a) was employed. |
| Refolding Assay | photoaffinity labeling |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |