Refolding Record:
| Protein | |
|---|---|
| Protein Name | Phospholipase A2, ammodytoxin A |
| Abbreviated Name | ATXA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Vipera ammodytes ammodytes (Western sand viper) |
| UniProt Accession | P00626 |
| 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 | 122 |
| Molecular Weight | 13788.8 |
| Pi | 8.35 |
| Molecular Weight | 13788.8 |
| Disulphides | 7 |
| Full Sequence |
SLLEFGMMILGETGKNPLTSYSFYGCYCGVGGKGTPKDATDRCCFVHDCCYGNLPDCSPKTDRYKYHRENGAIVCGKGTSCENRICECDRAAAICFRKNLKTYNYIYRNYPDFLCKKESEKC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Liang NS, Pungercar J, Krizaj I, Strukelj B, Gubensek F. (1993) FEBS Letters, 334, 55-59 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109 |
| Expression Temp | 37.0 |
| Expression Time | 8-10 h |
| Expression Vector | pMAXA |
| Expression Protocol | Expression of the fusion protein Routinely, a single fresh bacterial colony was picked from a M9 plate supplemented with 50 ,ug/ml ampicillin, inoculated into 2.5 ml of M9 medium with ampicillin and grown overnight at 37°C. The overnight culture was used to inoculate 250 ml of LB medium containing 100 ,ug/ml ampicillin in a 2 1 Erlemeyer flask. Bacterial culture was grown by shaking at 250 rpm and 37°C for 8 10 h to yield at the end about 2.2 g wet weight bacteria per liter of culture medium. If IPTG was used for induction, it was added at the final concentration of 0.4 mM when the cell density had reached A600 = 0.6. Cells were harvested by centrifugation at 4000 x g for 10 min. Inclusion bodies were isolated according to the procedure of Marston et al. [10]. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 2 M urea, 1% Triton X-100 and 5 mM DTT, and sulfonated with NTSB in 6 M GdnHCI, 0.3 M Na2SO3, pH 8.5 |
| Solubilization Buffer | 6 M GdnHC1, 50 mM Tris-HC1, pH 8.0, 10 mM CaC12, 3 mM EDTA and 2 mM reduced glutathion |
| Refolding Buffer | 0.3 M GdnHCI, 50 mM Tris-HC1, pH 8.0, 10 mM CaCI2, 3 mM EDTA, 2 mM reduced and 1 mM oxidized glutathione |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 48 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/1 mM |
| Refolding Protocol | Refolding, activation and purification of the recombinant protein Inclusion body pellet recovered from 2 1 of bacterial culture was thoroughly washed with a solution containing 2 M urea, 1% Triton X-100 and 5 mM DTT, and sulfonated with NTSB in 6 M GdnHCI, 0.3 M Na2SO3, pH 8.5 [11]. The fusion protein was precipitated by 1% acetic acid and dissolved in 100 ml of 6 M GdnHC1, 50 mM Tris-HC1, pH 8.0, 10 mM CaC12, 3 mM EDTA and 2 mM reduced glutathione. Refolding was performed by dilution 1:10 into a solution of 0.3 M GdnHCI, 50 mM Tris-HC1, pH 8.0, 10 mM CaCI2, 3 mM EDTA, 2 mM reduced and 1 mM oxidized glutathione. After standing for about 48 h at room temperature, the reoxidation mixture was concentrated to 100 ml by ultracentrifugation through a YM-10 membrane (Ami- con, USA) and trypsin was added to the final concentration of 4% by weight according to the fusion protein. Production of active ammodytoxin A was followed by measurement of PLA2 activity. After 2.5 h of stirring at room temperature, the pH was adjusted to 5.0, supernatant concentrated by ultrafiltration and dialysed against 50 mM sodium acetate, pH 5.0, 1 mM EDTA. This solution was loaded onto a Mono-S HR 5/5 FPLC column (Pharmacia) and purified with a linear gradient to 50 mM sodium acetate, pH 5.0, 1 mM EDTA and 2 M NaCI. Recombinant, more than 95% pure ammodytoxin A eluted as a sharp peak at about 0.6 M NaCI. The protein solution was desalted using a Centricon-10 microconcentrator (Amicon). |
| Refolding Assay | SDS-PAGE,enzyme activity,Toxicity testing |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 0.5 mg/L |
| Purity | 95% |
| Notes | n/a |