Refolding Record:
| Protein | |
|---|---|
| Protein Name | Decorin |
| Abbreviated Name | Decorin |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P21793 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 365 |
| Molecular Weight | 39879.1 |
| Pi | 8.72292 |
| Molecular Weight | 39879.1 |
| Disulphides | 0 |
| Full Sequence |
MKATIIFLLVAQVSWAGPFQQKGLFDFMLEDEASGIGPEEHFPEVPEIEPMGPVCPFRCQ
CHLRVVQCSDLGLEKVPKDLPPDTALLDLQNNKITEIKDGDFKNLKNLHTLILINNKISK
ISPGAFAPLVKLERLYLSKNQLKELPEKMPKTLQELRVHENEITKVRKSVFNGLNQMIVV
ELGTNPLKSSGIENGAFQGMKKLSYIRIADTNITTIPQGLPPSLTELHLDGNKITKVDAA
SLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLNNNKLVKVPGGLADHKYIQVVYL
HNNNISAIGSNDFCPPGYNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRAAVQLGNYK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hering TM, Kollar J, Huynh TD, Varelas JB. (1996) Analytical Biochemistry, 240, 98-108 |
| Project Aim | Structure-Function |
| Fusion | N-terminal maltose binding protein |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XL1-Blue |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pMAL-c |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 20 mM Tris/HCl, pH 7.4, 0.2 M NaCl, 1 mM DTT, and protease inhibitors (1 mM EDTA, 0.1 mM PMSF, 0.5 mg/liter leupeptin, 0.6 mg/liter aprotinin, 0.7 mg/liter pepstatin) |
| Solubilization Buffer | 50mM sodium acetate, 4M guanidinium chloride, 1mM DTT, protease inhibitors, pH 5.8 |
| Refolding Buffer | 50mM Tris-HCl, 0.2M NaCl, 1M guanidinium chloride, 1.25mM GSSG, 0.25mM GSH |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.3mg/ml |
| Refolding Time | 16h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | For refolding and final purification, a 12.5-ml aliquot of dissolved inclusion body protein was thawed and stirred at 47C. Using a 50-ml syringe fitted with a stopcock valve and needle, the following volumes of the indicated solutions were added by slow drip into the flask over the indicated time period with stirring: 12.5 ml of 2 M GdnHCl, 0.1 M NaCl, 0.05 M Tris/HCl, pH 9.0, over a 90-min period; 25 ml of a 1 M GdnHCl, 0.25 M NaCl, 0.05 M Tris/HCl, pH 9.0, over a 60-min period; and 50 ml of 0.3 M NaCl, 0.05 M Tris/HCl, pH 9.0, over a 120-min period. A final protein concentration of approximately 300 mg/ml in a total volume of 625 ml was achieved by adding at once 525 ml of 1 M GdnHCl, 0.2 M NaCl, 0.05 M Tris/HCl, pH 9.0. Proteinase inhibitors were added, and the pH was adjusted to 9.0. Reduced and oxidized glutathione were added to concentrationsof 1.25 and 0.25 mM, respectively, and this solution, which permits disulfides to reshuffle (51), was stirred overnight at 47C. The pH was readjusted to 7.2 and the solution was transferred to large diameter dialysis tubing, and dialyzed at 47C against 4 liters of 20 mM Tris/HCl, pH 7.2, 0.5 M NaCl, 1 mM EDTA, and protease inhibitors for 4 h initially, followed by two more changes of the same dialysis buffer (4 liters each) over the next 48 h. The protein was subsequently purified on amylose resin and the MBP tag cleaved with factor Xa. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 1-2mg/L culture |
| Purity | |
| Notes | |