Refolding Record:
| Protein | |
|---|---|
| Protein Name | Dihydrofolate reductase-thymidylate synthase |
| Abbreviated Name | TS-DHFR |
| SCOP Family | Dihydrofolate reductases |
| Structure Notes | |
| Organism | Plasmodium falciparum |
| UniProt Accession | P13922 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Dihydrofolate reductase domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 233 |
| Molecular Weight | 27103.5 |
| Pi | 8.81799 |
| Molecular Weight | 27103.5 |
| Disulphides | 0 |
| Full Sequence |
MMEQVCDVFDIYA ICACCKVESK NEGKKNEVFN NYTFRGLGNK GVLPWKCXSL DMKYFXAVTT YVNESKYEKL KYKRCKYLNK ETVDNVNDMP NSKKLQNVVV MGRTXWESIP KKFKPLSNRI NVILSRTLKK EDFDEDVYII NKVEDLIVLL GKLNYYKCFI IGGSVVYQEF LEKKLIKKIY FTRINSTYEC DVFFPEINEN EYQIISVSDV YTSNNTTLDF IIYKKTNNKM
|
| Notes | UniProt for Dihydrofolate reductase domain only: Q9GN27_PLAFA |
| Expression | |
|---|---|
| Report | Sirawaraporn W, Prapunwattana P, Sirawaraporn R, Yuthavong Y, Santi DV (1993) J Biol Chem, 268, 21637-21644 |
| 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)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET17b |
| Expression Protocol | Cells were grown in LB medium with 50microg/ml thymine and 100microg/ml ampicillin at 37degC until A600 reached 0.5-0.6. IPTG was added to 0.4mM final concentration and cells were grown for a further 3 hours before centrifugation (10000g, 10min, 4degC). The cell pellets were washed with 250ml cold phosphate-buffered saline (Ca2+ and Mg2+ free). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5-0.6 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM potassium phosphate pH 7.0, 0.1mM EDTA, 10mM DTT, 0.2M KCl, 6M GdnHCl |
| Refolding Buffer | 20mM potassium phosphate pH 7.0, 0.1mM EDTA, 10mM DTT, 0.2M KCl, 20% glycerol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 3h |
| Redox Agent | DTT |
| Redox Agent Concentration | 10mM |
| Refolding Protocol | Inclusion bodies were partially purified then dissolved in 10ml of solubilization buffer at 4degC. The mixture was stirred slowly at 4degC for at least 1h. Refolding was then performed by drop-wise, 20-fold dilution of the denatured proteins into refolding buffer. After 3h, the aggregates were pelleted centrifugation (10000g, 20min) and the soluble protein was then purified further using an MTX-sepharose CL-6B column |
| Refolding Assay | enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | 5-6mg/L culture |
| Purity | 80% |
| Notes | |