Refolding Record:
| Protein | |
|---|---|
| Protein Name | ProCathepsin D |
| Abbreviated Name | ProCatD |
| SCOP Family | Pepsin-like proteases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07339 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 393 |
| Molecular Weight | 42673.9 |
| Pi | 6.12164 |
| Molecular Weight | 42673.9 |
| Disulphides | 4 |
| Full Sequence |
A LVRIPLHKFT SIRRTMSEVG GSVEDLIAKG PVSKYSQAVP AVTEGPIPEV LKNYMDAQYY GEIGIGTPPQ CFTVVFDTGS SNLWVPSIHC KLLDIACWIH HKYNSDKSST YVKNGTSFDI HYGSGSLSGY LSQDTVSVPC QSASSASALG GVKVERQVFG EATKQPGITF IAAKFDGILG MAYPRISVNN VLPVFDNLMQ QKLVDQNIFS FYLSRDPDAQ PGGELMLGGT DSKYYKGSLS YLNVTRKAYW QVHLDQVEVA SGLTLCKEGC EAIVDTGTSL MVGPVDEVRE LQKAIGAVPL IQGEYMIPCE KVSTLPAITL KLGGKGYKLS PEDYTLKVSQ AGKTLCLSGF MGMDIPPPSG PLWILGDVFI GRYYTVFDRD NNRVGFAEAA RL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Conner GE, Richo G (1992) Biochemistry, 31, 1142-1177 |
| Project Aim | Structure-Function |
| 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 | not stated |
| Expression Vector | pTCPSD2 |
| Expression Protocol | Cells were grown in LB broth. not further details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Osmotic shock |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris pH 8.0 |
| Solubilization Buffer | 8M urea, 10mM Na-CAPS, pH 10.7, 50mM 2-mercaptoethanol |
| Refolding Buffer | 0.08M urea, 0.1mM Na-CAPS, 0.5mM 2-mercaptoethanol, pH 8.7 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.7 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.02mg/ml |
| Refolding Time | 2h |
| Redox Agent | GSH |
| Redox Agent Concentration | 5mM |
| Refolding Protocol | Harvested cells were resupsended in 20% sucrose, 50mM TrisHCl pH 8 and treated with 1mg/ml lysozyme for 10min at 4degC. After addition of 5mM EDTA pH 8.0, the incubation was continued for 20min at 4degC and 1h at 37degC. Bacteria were then lysed by the addition of 3 volumes of water. The lysate was adjusted to 5mM MgCl2 and digested with 3microg/ml DNaseI for 30min at room temperature. The suspension was centrifuged (10min, 15000g) and the inclusion bodies were washed 3 times by resuspension in was buffer. After the final wash, the inclusion bodies were resuspended in 10mM Tris pH 8.0, 0.1M EDTA and stored at -70degC. Inclusion bodies were dissolved in solubilization buffer and incubated for 30min at room temperature. The mixture was the centrifuged and the supernatant was diluted 100-fold in water. The diluted material was adjusted to pH 8.7 with HCl and after 2h was titrated to 3.7 with formic acid. After 24h, 5mM oxidized glutathione was added and the incubation was continued for various amounts of time (up to 7 days) |
| Refolding Assay | Enzyme activity,Sequence Analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |