Refolding Record:
| Protein | |
|---|---|
| Protein Name | D-ornithine aminomutase E component |
| Abbreviated Name | OraE |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Clostridium sticklandii |
| UniProt Accession | Q8VPJ5 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Heterodimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 755 |
| Molecular Weight | 83049.8 |
| Pi | 5.14185 |
| Molecular Weight | 83049.8 |
| Disulphides | 0 |
| Full Sequence |
MEKDLQLRVNEKLDVENILKDLDKYTPKRRGWTWRQPAENLQMGPFIYKDASTPLENSVA
LPSAKYFGDIDPQPLPVITTEIASGRFEDDIRRMRMAAWHGADHIMVIRTAGQSHYDGLI
EGTPQGIGGVPITRKQVRAQRKALDLIEEEVGRPINYHSYVSGVAGPDIAVMFAEEGVNG
AHQDPQYNVLYRNINMIRSFIDACESKTIMAWADMAQIDGIDGAHNANATAREAWKVMPE
LMVQHALNSIFSLKVGMKKSNICLSTVPPTAPPAPSMYLDLPYAVALREMFEGYRMRAQM
NTKYMEASTREATVTHVLNLLISKLTRADIQSTITPDEGRNVPWHIYNIEACDTAKQALI
GMDGLMDMVQLKREGVLGDTVRELKERAVLFMEEIIEAGGYFNAVEQGFFVDSGYYPERN
GDGIARQINGGIGAGTVFERDEDYMAPVTAHFGYNNVKQYDEALVSEPSKLIDGCTLEVP
EKIVYIDELDENDNVNVRMEETKEFRHSSMIKPEVEWQADGTVLLTMFLPTSKRVAEFAA
IEFAKKMNLEEVEVINREVMQEAEGTRIELKGRVPFSIDINSLVIPPEPEILSEDEIRED
IEKTPLKIVAATVGEDEHSVGLREVIDIKHGGIEKYGVEVHYLGTSVPVEKLVDAAIELK
ADAILASTIISHDDIHYKNMKRIHELAVEKGIRDKIMIGCGGTQVTPEVAVKQGVDAGFG
RGSKGIHVATFLVKKRREMREGK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chen HP, Wu SH, Lin YL, Chen CM, Tsay SS. (2001) J Biol Chem, 276, 44744-44750 |
| Project Aim | Functional Studies |
| 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 | unknown |
| Expression Vector | pET28a |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50 mM potassium phosphate buffer, 100 mM NaCl, 1 mM DTT, and 1 mM EDTA, 0.5% Triton X-100, pH 7.0 |
| Solubilization Buffer | 0.1 M potassium phosphate buffer, 6 M guanidine hydrochloride, 10 mM DTT, and 1 mM EDTA, pH 7.0 |
| Refolding Buffer | 0.1 M potassium phosphate buffer, 10% glycerol, and 1 mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 16h |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The cells were ruptured in a total volume of 30 ml by sonication. The insoluble fraction was collected by centrifugation at 25,000 ?g for 15 min, and the supernatant was discarded. The pellet was washed twice with 25 ml of the same buffer supplemented with 0.5% Triton X-100 to remove lipophilic contaminants. Each wash was followed by centrifugation at 25,000 ?g for 15 min. The pellet was solubilized in 25 ml of 0.1 M potassium phosphate buffer, pH 7.0, containing 6 M guanidine hydrochloride, 10 mM DTT, and 1 mM EDTA. The solution was stirred at room temperature for 2 h and cleared by centrifugation at 25,000 ?g for 15 min. After this, the purity of the OraE was better than 90% as judged by SDS-PAGE. No steps were taken to further purify OraE before refolding. The supernatant was used as the stock solution for refolding experiments and stored at 70 °C. The protein concentration of OraE in the stock solution was estimated from a Coomassie Blue-stained 10% SDS-polyacrylamide gel. All steps were performed at 4 °C or on ice. About 3.8 mg of OraS in 2 ml of 50% glycerol and 9.1 mg of OraE in 3-ml stock solution were added dropwise to 100 ml of 0.1 M potassium phosphate buffer, pH 7.0, containing 1 M guanidine hydrochloride, 10% glycerol, and 1 mM DTT, with gentle stirring. After the solution was stirred for a further 20 min, about 400 µl of 12 mM PLP or 100 µl of 2 mM adenosylcobalamin was added. The solution was then dialyzed overnight against 3 ?2 liters of 0.1 M potassium phosphate buffer, pH 7.0, containing 10% glycerol and 1 mM DTT in the dark. The dialysate was then cleared by centrifugation at 25,000 ?g for 15 min and concentrated to 20 ml by ultrafiltration in a stirred cell fitted with a YM 3 membrane. To remove excess OraS and other contaminating components, the refolded enzyme was then loaded onto a Q-Sepharose HP anion-exchange column (2.6 ?20 cm), equilibrated in 10 mM potassium phosphate buffer, pH 7.0, which contained 10% glycerol and 1 mM DTT. The column was first washed with 50 ml of the same buffer, and proteins were then eluted with a 500-ml linear gradient of 0-0.5 M KCl. The flow rate was 2 ml/min; 6-ml fractions were collected. Active fractions were pooled and concentrated to 8 ml by ultrafiltration as described above. The protein solution was stored at 20 °C in the presence of 50% glycerol. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | >90% |
| Notes | |