Refolding Record:
| Protein | |
|---|---|
| Protein Name | Magnesium chelatase D subunit |
| Abbreviated Name | BchD |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rhodobacter sphaeroides |
| UniProt Accession | O34845 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 599 |
| Molecular Weight | 62836.1 |
| Pi | 8.73072 |
| Molecular Weight | 62836.1 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHM MPLGPWERVE AALTLLAIDP AGLKGLWLRA RASALRDRIT GALGALPLPV RRIHPTIGDD ALFGGLDLAA TLSAGTPVVQ KGILDEPAVL VLAMAERTLP
GLAARLGTAL DAPRHCLIAL DEGAERDELL PLGLVDRLAL FLDLDGLPWG ETREIALDPE RLAAARARLA AVATPPEAAA TLARVAAQLG IASLRAPTLA LAAARAQAAW EGHAAVTDED IRRAADLVFA HRAMPASEEA PPEPEPEPPE DQPDDSPPPP EQQQGEEMFP EEMLVEAVRA ALPADLLEQL AAGRAARMAR
GATGTGSAKA GNRRGRPLPS RMGRLGTGAR IDLVGTLRAA APWQPLRRRQ QKTDAVLLVR PSDIRIKRFR ETSDRVLIFA VDASGSSAMA RLSEAKGAVE LLLGQAYARR DHVSLLAFRG RDAELILPPT RSLVQTKRRL AGLPGGGGTP LAHGLRLALA VGLQARARGM TPTVALLTDG RGNIALDGSA NRAQAEEDAL KLAASLRGSG LPAVVIDTAN RPQPSLAALA RALDAPYIAL PRADAHKLSN VLGAAMGD APAANKARKEAELAAATAEQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gibson LCD, Jensen PE, Hunter CN (1999) Biochem J., 337, 243-251 |
| Project Aim | Structure-Function |
| Fusion | N-terminal hexahistidine tag |
| 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 | 4h |
| Expression Vector | pET14b |
| Expression Protocol | Cells were grown at 37deg C in 100ml LB medium containing 100mg/mL ampicillin until A600 reached 0.6-1. Protein expression was induced with 0.4mM IPTG and cells were incubated a further 4h before centrifugation and storage at -20degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-1 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution with complex partner subunits |
| Wash Buffer | n/a |
| Solubilization Buffer | 5mM imidazole, 500mM NaCl, 20mM Tris/HCl pH 7.0, 6M urea |
| Refolding Buffer | 50mM Tricine pH 7.9 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.9 |
| Refolding Temperature | 34.0 |
| Protein Concentration | |
| Refolding Time | 5min |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cell pellet from 1L culture was thawed at room temperature and resuspended in solubilization buffer, incubated on ice for 1hr and then centrifuged (100000g, 1hr). The supernatant was loaded onto a Ni2+-agarose affinity column. The protein was eluted with solubilization buffer containing 500mM imidazole. The protein was then dialyzed against 50mM Tricine/NaOH pH 7.9, 6M urea and stored at 4degC. For refolding, 100microL of the purified protein (typically 8microg) was diluted with 900microL of refolding buffer containing BchI (typically 20microg), MgCl2 and ATP, so that the final concentration of MgCl2 and ATP were 20mM and 4mM respectively. The mixture was incubated at 34degC for 5min. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 80mg/L |
| Purity | |
| Notes | Purification of BchD from soluble fraction yielded 2mg/L (compared with 80mg/L from insoluble fraction). BchD could not be refolded on its own, but refolded when directly added to Mg chelatase assay (with BchI). |