Refolding Record:
| Protein | |
|---|---|
| Protein Name | General odorant-binding protein 1 |
| Abbreviated Name | GOBP1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Manduca sexta (Tobacco hawkmoth) (Tobacco hornworm |
| UniProt Accession | P31418 |
| 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 | 145 |
| Molecular Weight | 16950.4 |
| Pi | 5.22 |
| Molecular Weight | 16950.4 |
| Disulphides | 3 |
| Full Sequence |
DVQVMKDVTLGFGQALEQCREESQLTEEKMEEFFHFWREDFKFEHRELGCALQCMSRHFNLLTDSSRMHHENTDKFIKSFPNGAVLSKTMVELIHNCELQHDAEEDHCWRILRVAECFKISCTKAGIAPSMEVMMAEFIMELKQ
|
| 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 (4000g), dispersed in lysis buffer (80 mm Tris-HC1, RECOMBINANT GOBP 407 200 mm NaC1, 1 mm ethylene diamine tetraacetic acid (EDTA), 4% glycerol, pH 7.2, 1 mM phenylmethyl sulfonyl fluoride (PMSF), lysed by French Press (14,000 psi, two passes), or sonication (10 s, five to six passes). Soluble proteins were isolated by ultracentrifugation (30,000 g) and ultrafiltered against nanopure water.in 10 mM Tris buffer pH 6.8 or 8.0 was prepared for the salt dependence study. |
| 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 |