Refolding Record:
| Protein | |
|---|---|
| Protein Name | Dihydrofolate Reductase |
| Abbreviated Name | DHFR |
| SCOP Family | Dihydrofolate reductases |
| Structure Notes | |
| Organism | Pneumocystis carinii |
| UniProt Accession | P16184 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 209 |
| Molecular Weight | 23883.5 |
| Pi | 9.1945 |
| Molecular Weight | 23883.5 |
| Disulphides | 0 |
| Full Sequence |
MNQQKSLTLIVALTTSYGIGRSNSLPWKLKKEISYFKRVTSFVPTFDSFESMNVVLMGRK
TWESIPLQFRPLKGRINVVITRNESLDLGNGIHSAKSLDHALELLYRTYGSESSVQINRI
FVIGGAQLYKAAMDHPKLDRIMATIIYKDIHCDVFFPLKFRDKEWSSVWKKEKHSDLESW
VGTKVPHGKINEDGFDYEFEMWTRDL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Delves CJ, Ballantine SP, Tansik RL, Baccanari DP, Stammers DK. (1993) Protein Expression and Purification, 4, 16-23 |
| 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 |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pT7-7 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris-HCl, 100mM NaCl, 1mM EDTA, 1mM DTT, 1M guanidinium chloride pH 8.0 |
| Solubilization Buffer | 50mM potassium phosphate, 2mM DTT, 2mM EDTA, 0.5% PEG 1450, 4M guanidine hydrochloride, pH 7.0 |
| Refolding Buffer | 50mM potassium phosphate, 2mM DTT, 2mM EDTA, 0.5% PEG 1450 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.2mg/ml |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were washed twice with wash buffer. Refolding was optimal at 0.2mg/ml protein concentration and was achieved by dialysis. The refolded protein was purified further by methotrexate sepharose chromatography. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 50%- 6mg/L culture |
| Purity | 90% |
| Notes | |