Refolding Record:
| Protein | |
|---|---|
| Protein Name | Nitrile hydratase |
| Abbreviated Name | NHase |
| SCOP Family | Nitrile hydratase beta chain |
| Structure Notes | |
| Organism | Rhodococcus sp. |
| UniProt Accession | Q59786 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 203 |
| Molecular Weight | 22790.7 |
| Pi | 4.83 |
| Molecular Weight | 22790.7 |
| Disulphides | Unknown |
| Full Sequence |
MSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADPRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEDELAKLVSRDSMIGVSNALTPQEVIV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ikehata O, Nishiyama M, Horinouchi S, Beppu T. (1998) Eur J Biochem., 181, 563-570 |
| Project Aim | Structural Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM105 |
| Expression Temp | 30.0 |
| Expression Time | 20 h |
| Expression Vector | pyuk121 |
| Expression Protocol | E. coli JM105 containing pYUK121, which will be described later, was grown at 30°C overnight in 10 ml 2 x YT medium containing 10 mg/l FeS04 . 7 H20, 1 pg/l PQQ and 50 pg/ml ampicillin and transferred to 1 1 of the same medium. After a 2-h incubation at 30 \"C, isopropyl p-D-thiogalactopyranoside (IPTG) was added at a final concentration of 2 mM to induce the lac promoter and portions (100 ml each) were taken at 5 h, 10 h, 15 h and 20 h after the addition of IPTG. Cells were collected by centrifugation, suspended in 3 ml 50 mM phosphate buffer (pH 7.7) containing 0.35% sodium n-butyrate, and disrupted by sonication. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 ml 50 mM phosphate buffer (pH 7.7) containing 0.35% sodium n-butyrate, 8 M urea, 10 mg/l FeS04. 7H20 and 1 pg/l PQQ |
| Refolding Buffer | 3 1 50 mM Tris/HCl (pH 10.0) containing 0.35% sodium n-butyrate, and then against 3 1 50 mM phosphate buffer (pH 7.7) containing 0.35% sodium n-butyrate, 10 mg/l FeS04 . 7 H20 and 1 pg/l PQQ |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 10.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | 18 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The sonicates were then centrifuged at 25000 x g for 30 min. The super- natant and the pellet were used for the nitrile hydratase assay. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was performed according to the method of Laemmli [20]. For activation of the NHase, the pellet thus obtained was dissolved in 20 ml 50 mM phosphate buffer (pH 7.7) containing 0.35% sodium n-butyrate, 8 M urea, 10 mg/l FeS04 . 7H20 and 1 pg/l PQQ. The solution was then centrifuged at 40000 x g for 1 h. Sodium chloride (0.585 g) was added to the supernatant and the solution was adjusted to pH 10.0 with 1 M NaOH. The solution was dialyzed first against 3 1 50 mM Tris/HCl (pH 10.0) containing 0.35% sodium n-butyrate, 10mg/l FeS04 . 7H20 and 1 pg/l PQQ for 3 h and then against 3 1 50 mM phosphate buffer (pH 7.7) containing 0.35% sodium n-butyrate, 10 mg/l FeS04 . 7 H20 and 1 pg/l PQQ for 15 h. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |