Refolding Record:
| Protein | |
|---|---|
| Protein Name | Phospholipase A2 |
| Abbreviated Name | PLA2 |
| SCOP Family | Vertebrate phospholipase A2 |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P04054 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 126 |
| Molecular Weight | 14139.0 |
| Pi | 7.94 |
| Molecular Weight | 14139.0 |
| Disulphides | Unknown |
| Full Sequence |
AVWQFRKMIKCVIPGSDPFLEYNNYGCYCGLGGSGTPVDELDKCCQTHDNCYDQAKKLDSCKFLLDNPYTHTYSYSCSGSAITCSSKNKECEAFICNCDRNAAICFSKAPYNKAHKNLDTKKYCQS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Han SK, Yoon ET, Cho W. (1998) Biochem J., 331, 353-357 |
| 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 | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET21a |
| Expression Protocol | Expression and purification of hV-PLA2 Escherichia coli strain BL21(DE3) was used as a host for the protein expression. Luria broth (4 l) containing 100 µg\\ml ampicillin was inoculated with 40 ml of overnight culture from a freshly transformed single colony ; the culture was grown at 37 mC. When the absorbance of the culture at 600 nm reached 0.2, additional ampicillin was added to a final concentration of 1 mM. The culture was induced by the addition of 0.5 mM IPTG when the absorbance of the culture at 600 nm was 0.8. After the culture was incubated for 4 h at 37 mC, cells were harvested at 3000 g for 10 min at 4 mC and frozen at k20 mC. The cells were resuspended in 100 ml of 0.1 M Tris\\HCl buffer, pH 8.0, containing 5 mM EDTA, 50 mM NaCl, 0.5 mM PMSF, 0.5 % (v\\v) Triton X-100 and 0.4 % (w\\v) sodium deoxycholate, and stirred at 4 mC. After cells were disrupted by sonication, the inclusion body pellet was obtained by centrifugation at 10 000 g for 20 min |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50 ml of 50 mM Tris\\HCl, pH 8.0, containing 5 M urea and 5 mM EDTA |
| Solubilization Buffer | 5 M guanidinium thiocyanate solution (pH 8.5) containing 0.3 M sodium sulphite |
| Refolding Buffer | 80 ml of 50 mM Tris\\HCl, pH 7.4, containing 10 % (v\\v) glycerol, 8 mM reduced glutathione and 7 mM oxidized glutathione |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 20 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 8/7 mM |
| Refolding Protocol | The pellet was resuspended and washed in 100 ml of 0.8 % (v\\v) Triton X-100\\0.8 % (w\\v) sodium deoxycholate (i2). The pellet was collected by centrifugation and washed in 50 ml of 50 mM Tris\\HCl, pH 8.0, containing 5 M urea and 5 mM EDTA, and centrifuged. Inclusion bodies were solubilized in 10 ml of 5 M guanidinium thiocyanate solution (pH 8.5) containing 0.3 M sodium sulphite, and stirred vigorously at room temperature for 30 min. 2-Nitro-5-(sulphothio)-benzoate solution (4 ml ; 50 mM) was then added, and the modification was monitored spectrophotometrically at 412 nm. After the modification was complete ( 20 min), any insoluble matter was removed by centrifugation at 35 000 g for 15 min at room temperature. The reaction mixture was loaded on to a Sephadex G-75 column (2.5 cmi35 cm) equilibrated with 25 mM Tris\\HCl buffer, pH 8.0, containing 5 M urea and 5 mM EDTA, and the first major retained peak was collected (80 ml) and dialysed against water and then against 0.3 % (v\\v) acetic acid to precipitate the sulphonated protein. The precipitated protein was washed with 100 ml of water and resuspended in 10 ml of 50 mM Tris\\HCl buffer, pH 7.4, con- taining 5 mM EDTA and 8 M guanidinium chloride. To this solution of sulphonated protein, 80 ml of 50 mM Tris\\HCl, pH 7.4, containing 10 % (v\\v) glycerol, 8 mM reduced glutathione and 7 mM oxidized glutathione was added dropwise over 3 h. The solution was kept at room temperature for 20 h, at which point protein solution was dialysed overnight against 4 l of 25 mM Tris\\HCl buffer containing 0.2 M guanidinium chloride and 10 % (v\\v) glycerol, pH 7.4 (i2). The clear solution was loaded on to a HiLoad 16\\10 S Sepharose column (Pharmacia) attached to an FPLC system (Pharmacia). The folded protein was eluted with a gradient of NaCl from 0 to 0.5 M in 25 mM Tris buffer containing 0.2 M guanidinium chloride and 10 % (v\\v) glycerol, pH 7.4. The major protein peak was collected, and stored at k4 mC. The purity of the protein was confirmed by SDS\\PAGE. Protein concentration was determined by the bicinchoninic acid method (Pierce). The N-terminal amino acid sequence of recombinant hV-PLA2 was determined by Edman degradation using an Applied Biosystems 477A Protein Sequencer. Fragments were sequentially separated with reverse-phase HPLC and compared with standards. |
| Refolding Assay | Kinetic analysis |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | 0.3 mg /L |
| Purity | n/a |
| Notes | n/a |