Refolding Record:
| Protein | |
|---|---|
| Protein Name | Phospholipase A2 BA2 |
| Abbreviated Name | BAPLA2-II |
| SCOP Family | Vertebrate phospholipase A2 |
| Structure Notes | |
| Organism | Agkistrodon halys pallas (Chinese water mocassin) |
| UniProt Accession | O42190 |
| 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 | 124 |
| Molecular Weight | 13801.6 |
| Pi | 5.0 |
| Molecular Weight | 13801.6 |
| Disulphides | 7 |
| Full Sequence |
NLLQFEKMIKKMTGKEPVVSYAFYGCYCGSGGQGKPKDATDRCCFVHDCCYGKVTGCDPKMDVYSFSEENGDIVCGGDDPCKKEICECDRAAAICFRDNLNTYNDKKYWAFGAKNCPQEESEAC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Liu X, Pan H, Yang G, Wu X, Zhou Y. (1999) Biochemica et Biophysica Acta, 1431, 157-165 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | RRI |
| Expression Temp | 42.0 |
| Expression Time | 4 h |
| Expression Vector | pBLMVL2 |
| Expression Protocol | Expression of cDNA The assembled gene was subcloned into the expression vector pBLMVL2 and transferred into E. coli (RR1). Bacterial cultures were grown in fresh LB medium at 32°C. When the A595 reached 0.6, the temperature was raised to 42°C and the cells incubated for 4 h. Cells were harvested by centrifugation (5000×g), cell pellets were suspended (1 ml/50 ml culture) in 50 mM Tris-HCl (pH 7.5) containing 25% sucrose and frozen at −70°C. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD 0.6 = 595 |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM Tris-HCl, pH 8.5, 10 mM DTT, 5 mM EDTA |
| Solubilization Buffer | 8 M guanidine-HCl and 10 mM DTT |
| Refolding Buffer | 2 M urea, 4 mM EDTA, 0.1 M NH4Cl, 20 mM sodium borate (pH 8.3) |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.3 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 4 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Denaturation, refolding and purification Purification of insoluble inclusion bodies was done according to the method of Kelley [21]. After thawing the cells on ice, lysozyme was added to a concentration of 0.5 mg/ml, EDTA to 10 mM, DTT to 10 mM, NaCl to 100 mM, and the mixture was chilled on ice for 1 h. Cells were lysed by four freeze/thaw cycles from a CO2/ethanol bath to a 37°C water bath. RNase A and DNase I were added to a final concentration of 10 μg/ml and MgCl2 to 5 mM. After a 30-min incubation at room temperature, the mixture was centrifuged at 13 000×g for 10 min at 4°C. The pellet containing insoluble recombinant PLA2 protein (inclusion bodies) was first washed with buffer (50 mM Tris-HCl, pH 8.5, 10 mM DTT, 5 mM EDTA) and then with buffer containing 1% Triton X-100 and 10 mM EDTA. The pellet was resuspended in buffer (1 ml/50 ml starting culture) containing 8 M guanidine-HCl and 10 mM DTT and solubilized overnight at 4°C. Refolding was done according to the method of Dulder et al. [22]. The solution of denatured protein was dialyzed three times against 1 l of freshly made 2 M urea, 4 mM EDTA, 0.1 M NH4Cl, 20 mM sodium borate (pH 8.3) with 4 h before changes to remove guanidine-HCl and DTT. To the fourth change of dialysis solution was added cysteine to 8 mM and cystine to 1 mM and the dialysate was placed in the dark. Aliquots were tested every 5 h for PLA2 enzymatic activity. When maximal activity was reached the protein solution was again dialyzed overnight against 5 mM HAc at 4°C and lyophilized. Finally, FPLC column Superose 12 was used to isolate BAPLA2-II from the refolding mixture. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |