Refolding Record:
| Protein | |
|---|---|
| Protein Name | Dihydroorotate dehydrogenase |
| Abbreviated Name | PyrDII |
| SCOP Family | Ferredoxin reductase FAD-binding domain-like |
| Structure Notes | |
| Organism | Bacillus subtilis |
| UniProt Accession | PYRK_BACU |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 256 |
| Molecular Weight | 28099.5 |
| Pi | 5.68339 |
| Molecular Weight | 28099.5 |
| Disulphides | 0 |
| Full Sequence |
MKKAYLTVCS NQQIADRVFQ MVLKGELVQG FTTPGQFLHL KVSEAVTPLL RRPISIADVN FEKNEVTIIY RVDGEGTRLL SLKQQGELVD VLGPLGNGFP VNEVQPGKTA LLVGGGVGVP PLQELSKRLI EKGVNVIHVL GFQSAKDVFY EEECRQYGDT YVATADGSYG ETGFVTDVIK RKKLEFDILL SCGPTPMLKA LKQEYAHKEV YLSMEERMGC GIGACFACVC HTNESETSYV KVCLDGPVFK AQEVAL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kahler AE, Nielsen FS, Switzer RL (1999) Archives Biochemistry and Biophysics, 371, 191-201s |
| 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 | pET20b |
| Expression Protocol | Cells were grown in LB medium containing 100microg/ml ampicillin at 37degC. Expression was induced with IPTG. Cells were harvested and resuspended in 50mM sodium phosphate buffer pH 6.0 |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 25mM TrisHCl pH 8.0, 100mM KCl, 12.5mM MgCl2, 0.5mM EDTA, 10% glycerol |
| Solubilization Buffer | 25mM TrisHCl pH 8.0, 100mM KCl, 12.5mM MgCl2, 0.5mM EDTA, 10% glycerol, 40mM DTT, 4M GdnHCl |
| Refolding Buffer | 25mM TrisHCl pH 8.0, 100mM KCl, 12.5mM MgCl2, 0.5mM EDTA, 10% glycerol OR 25mM TrisHCl 10% glycerole |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 3mg/ml |
| Refolding Time | 3h |
| Redox Agent | DTT |
| Redox Agent Concentration | 3mM,3mM |
| Refolding Protocol | 1L culture worth of cells was lysed by sonication on ice, then centrifuged (10min, 27000g). The cell pellet was washed in 30ml wash buffer three times. The cells were then emulsified by suspension in 2ml wash buffer containing 0.1% Triton X-100, then to this was added 1ml wash buffer without Triton X-100, 2ml of 200mM DTT and 5ml of 8M GdnHCl, the final concentrations of DTT and GdnHCl were 40mM and 4M respectively in 10ml. The protein was then dialysed against 100ml of solubilisation buffer -this dialysis step was then repeated. The sample was then transferred to 1L refolding buffer and dialyzed for 2h, this step was repeated once or twice with fresh buffer. |
| Refolding Assay | Ligand Binding,SDS-PAGE,Visible spectra |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | 31mg/L culture |
| Purity | n/a |
| Notes | Protein could be reconstituted by incubation with FAD or FMN cofactors (see paper for more details) |