Refolding Record:
| Protein | |
|---|---|
| Protein Name | Pheromone-binding protein |
| Abbreviated Name | PBP |
| SCOP Family | Insect pheromone/odorant-binding proteins |
| Structure Notes | |
| Organism | Antheraea polyphemus |
| UniProt Accession | P20797 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 143 |
| Molecular Weight | 15783.1 |
| Pi | 4.74 |
| Molecular Weight | 15783.1 |
| Disulphides | 3 |
| Full Sequence |
SPEIMKNLSNNFGKAMDQCKDELSLPDSVVADLYNFWKDDYVMTDRLAGCAINCLATKLDVVDPDGNLHHGNAKDFAMKHGADETMAQQLVDIIHGCEKSAPPNDDKCMKTIDVAMCFKKEIHKLNWVPNMDLVIGEVLAEV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Prestwich GD. (1993) Protein Science, 2, 420-428 |
| Project Aim | Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XA-90 |
| Expression Temp | 37.0 |
| Expression Time | 12 h |
| Expression Vector | GDP-Apo3c |
| Expression Protocol | Two ampicillin-resistant colonies were obtained. Each colony was grown in 3 mL of LB-ampicillin (LBA) medium for 12 h at 37 \\\"C and replated to obtain single clones. Cells grown to early log phase (Am = 0.4-0.6) were induced with 1 mM IPTG, and cells from both induced and uninduced cultures were isolated by centrifugation. Aliquots were analyzed for protein production and for the presence of the desired Apo-3 cassette insert. Thus, cells were lysed by sonication or by lysozyme-freeze-thaw methods (Sambrook et al., 1989), and soluble and insoluble proteins were analyzed by SDS-PAGE (see below). Plasmid DNA was isolated by the CTAB (cetyl trimethylammonium bromide) miniprep technique and digested as above with Eco RI and Hind 111. Inserts were visualized in 1% agarose gels containing ethidium bromide. Large scale fermentations were conducted in l-L flasks in LBA medium using starter cultures from overnight 10-mL incubations. After induction and protein synthesis (4-6 h, 37 \\\"C), cells were harvested in l-L bottles at 3,500 x g, lysed in a 80 mM Tris-HCI, 200 mM NaCI, 1 mM EDTA, 4% glycerol, pH 7.2, buffer containing 0.5 mM PMSF using a French press (14,000 psi, 4 \\\"C, 3-4 passes), and soluble proteins were isolated by ultracentrifugation (30,000 x g) followed by ultrafiltration with 20 mM Tris, pH 7.2, buffer. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4-0.6 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.2% Triton X-100 in 50 mM Tris buffer (pH 6.8) |
| Solubilization Buffer | 6 N guanidinium hydrochloride |
| Refolding Buffer | 5 mM cysteine in 100 mM Tris-HCI, pH 8.0 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 5 mM,5 mM |
| Refolding Protocol | Protein refolding Insoluble protein from cell lysates from a l-L 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. Two refolding protocols were attempted. The redox method (De Bernardez-Clark & Georgiou, 1991; Seeley & Young, 1991) was performed as follows. To 2.5 mL of solubilized inclusion body protein was added 2.5 mL of 10 mM DTT in 200 mM Tris-HC1 (pH 8.0). After reduction of the solubilized protein at room temperature for 30-60 min, 1 mL of 100 mM cystine (dissolved in 0.5 N NaOH) was added to give ca. 14 mM cyst(e)ine. After incubation for 10 min to oxidize remaining DTT, this mixture was added in portions to 10 volumes of 5 mM cysteine in 100 mM Tris-HCI, pH 8.0. The final mixture, containing 0.5-2.0 mg/mL protein, and ca. 5-6 mM cyst(e)ine was shaken in air on an orbital shaker for 24 h at room temperature. The precipitated, aggregated protein was removed by centrifugation (6,000 X g, 30 rnin), and the supernatant was dialyzed overnight against 200 mM Tris pH 8.5. The sample was concentrated and further desalted by ultrafiltration (YM10). The final concentration for ca. 50 mL was 1.5 mg/mL. This protocol was later repeated on a 4-L scale fermentation by a fourfold scaleup to obtain ca. 6 mL at 40 mg/mL. |
| Refolding Assay | Western Blot,photoaffinity labeling |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |