Refolding Record:
| Protein | |
|---|---|
| Protein Name | Polyhydroxyalkanote depolymerase |
| Abbreviated Name | phaZ |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Pseudomonas putida |
| UniProt Accession | Q6PLI2 |
| 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 | 283 |
| Molecular Weight | 31283.4 |
| Pi | 9.48 |
| Molecular Weight | 31283.4 |
| Disulphides | Unknown |
| Full Sequence |
MPQPYVFRTVELDDQSIRTAVRPGKPHLTPLLIFNGIGANLELVFPFIEALDPDLEVIAFDVPGVGGSSTPRQPYRFPGLAKLTARMLDYLDYGQVNVIGVSWGGALAQQFAHDYPERCKKLVLAATAAGAVMVPGKPKVLWMMASPRRYVQPSHVMRIAPLIYGGAFRRDPPLAARHAAKVRSGGKLGYYWQLFAGLGWTSIHWAAQDQPADSGLAGDDDPLIPLINMRLLAWRIPNAQLHIIDDGHLFLIIRAEAVAPIIMKFLEQERQRARMHPRPASGT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jiang Y, Ye J, Wu H, Zhang H. (2004) Biotechnol Lett, 26, 1585-1588 |
| Project Aim | Cloning and expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET11a |
| Expression Protocol | The plasmid pET-PHA was transformed into E. coli BL21 (DE3). The transformants were grown at 37 ◦ C in LB medium containing 100 µg ampicillin ml−1 . Expression production was induced with 1 mM IPTG until the culture turbidity at 600 nm was 0.5, and the cells were grown for an additional 4 h at 37 C. The cells were harvested by centrifugation at 6000 g for 10 min and lysed by ultrasonication. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM Tris/HCl, 1 mM EDTA, pH 8 |
| Solubilization Buffer | 20 mM Tris/HCl (pH 8) containing 8 M urea and 0.5% β -mercaptoethanol |
| Refolding Buffer | 20 mM Tris/HCl (pH 8), 1 mM GSH and 0.2 mM GSSG |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 20 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/0.2 mm |
| Refolding Protocol | The cells were harvested by centrifugation at 6000 g for 10 min and lysed by ultrasonication. Inclusion bodies were then separated by centrifugation at 15 000 g for 30 min at 4 ◦ C and purified with 0.5% (v/v) Triton buffer (containing 50 mM Tris/HCl, pH 8), then washed with TE buffer (20 mM Tris/HCl, 1 mM EDTA, pH 8) 3 times to remove the triton, and finally, solubilized in 20 mM Tris/HCl (pH 8) containing 8 M urea and 0.5% β -mercaptoethanol. The denatured depolymerases was rapidly diluted into folding buffer containing 20 mM Tris/HCl (pH 8), 1 mM GSH and 0.2 mM GSSG. The folding process was performed at 25 ◦ C for no less than 20 h. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |