Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alkaline phosphatase |
| Abbreviated Name | ALP |
| SCOP Family | Alkaline phosphatase |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P19111 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 495 |
| Molecular Weight | 52485.8 |
| Pi | 5.61 |
| Molecular Weight | 52485.8 |
| Disulphides | 2 |
| Full Sequence |
LVPVEEEDPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLG
DGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLC
GVKGNYRTIGVSAAARYNQCKTTRGNEVTSVMNRAKKAGKSVGVVTTTRVQHASPAGAYA
HTVNRNWYSDADLPADAQMNGCQDIAAQLVNNMDIDVILGGGRKYMFPVGTPDPEYPDDA
SVNGVRKRKQNLVQAWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQD
HTKDPTLQEMTEVALRVVSRNPRGFYLFVEGGRIDHGHHDDKAYMALTEAGMFDNAIAKA
NELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPSKALDSKSYTSILYGNGPGYAL
GGGSRPDVNDSTSEDPSYQQQAAVPQASETHGGEDVAVFARGPQAHLVHGVEEETFVAHI
MAFAGCVEPYTDCNLPAPTTATSIPD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Yazdanparast R, Khodagholia F, Sourib E (2007) International Journal of Biological Macromolecules, 1, 1 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 100 mM Tris–HCl buffer (pH 7) containing 8 M urea |
| Solubilization Buffer | 0.24 mg/ml ALP, 100 mM Tris–HCl pH 7, 0.8 M urea and 1.4 mM of the detergent |
| Refolding Buffer | 0.12 mg/ml ALP, 100 mM Tris–HCl pH 7, 0.4 M urea and 0.7 mM of each of the detergents |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 3 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Denatured ALP was prepared by incubating 2.4 mg/ml of the enzyme in 100 mM Tris–HCl buffer (pH 7) containing 8 M urea at 25 °C for 24 h. Protein denaturation was confirmed by activity determination as well as fluorescence measurements [28]. For protein refolding, the denatured ALP was diluted by a factor of 10 with Tris–HCl buffer containing the detergent to give concentrations of 0.24 mg/ml ALP, 100 mM Tris–HCl pH 7, 0.8 M urea and 1.4 mM of the detergent. After 5 min, the second detergent solution was added to bring the final concentrations to 0.12 mg/ml ALP, 100 mM Tris–HCl pH 7, 0.4 M urea and 0.7 mM of each of the detergents. For unassisted samples, the enzyme was diluted by a factor of 20 with Tris–HCl buffer to bring the final concentrations to 0.12 mg/ml ALP and 0.4 M urea. After 3 h incubation at room temperature, the enzymatic activities of the refolded samples were determined spectrophotometrically as will be provided in the next section. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |