Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cytochrome b6 |
| Abbreviated Name | petB |
| SCOP Family | Cytochrome b of cytochrome bc1 complex (Ubiquinol-cytochrome c reductase) |
| Structure Notes | |
| Organism | Spinacia oleracea |
| UniProt Accession | P00165 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 216 |
| Molecular Weight | 24166.6 |
| Pi | 8.88 |
| Molecular Weight | 24166.6 |
| Disulphides | Unknown |
| Full Sequence |
MSKVYDWFEERLEIQAIADDITSKYVPPHVNIFYCLGGITLTCFLVQVATGFAMTFYYRPTVTDAFASVQYIMTEVNFGWLIRSVHRWSASMMVLMMILHVFRVYLTGGFKKPRELTWVTGVVLGVLTASFGVTGYSLPWDQIGYWAVKIVTGVPDAIPVIGSPLVELLRGSASVGQSTLTRFYSLHTFVLPLLTAVFMLMHFLMIRKQGISGPL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Króliczewski J, Szczepaniak A. (2002) Biochemica et Biophysica Acta, 1598, 177-184 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | TB1 |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pMal-c2 |
| Expression Protocol | E. coli TB1 cells, transformed with pMal-c2b6, were grown aerobically at 37 °C in 1 l of Luria-Bertani medium containing 100 μg/ml ampicillin and inoculated with 10 ml overnight cultures. The production of fusion protein (MBP–apocytochrome b6) was induced by addition of 0.5 mM IPTG (isopropyl thio-β- -galactoside) at an OD550 of 0.6. After shaking for 4 h, cells were harvested by centrifugation (5000×g 15 min), resuspended in 30 ml of TEN buffer (20 mM Tris–HCl, pH 7.8, 10 mM EDTA, 1 mM PMSF, 100 mM NaCl) and then frozen overnight. After thawing, the solution was sonicated (6×15 s, 65 W microtip sonifier cell disrupter, Barnsted) and the lysate was centrifuged at 10 000×g, 20 min. The pellet fraction of inclusion bodies (IB), which were resuspended in 20 ml of buffer TEN supplemented with RNAse and DNAse (both 10 μg/ml, Sigma), was stirred at room temperature (RT) for 30 min and next centrifuged at 5000×g, 60 min, 4 °C. Insoluble materials were washed several times in TENT buffer (50 mM Tris–HCl, pH 7.8, 1 mM EDTA, 1 mM PMSF, 200 mM NaCl 2% v/v Triton X-100). Triton was removed by centrifugation (4300×g, 20 min), washed twice in TEN buffer (20 min shaking at 37 °C), and further centrifugation (5000×g, 30 min) was done, yielding a white pellet of IB contained MBP–apocytochrome b6 fusion protein. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 550 |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | TEN buffer (50 mM Tris–HCl, pH 7.8, 1 mM EDTA, 1 mM PMSF, 200 mM NaCl 2% v/v Triton X-100) |
| Solubilization Buffer | 7 M Urea in TEN buffer |
| Refolding Buffer | 50 mM Tris–HCl, pH 8.0, 50 mM NaCl, and 10 mM sodium dithionite |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 30 min |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The hemin was obtained from Fluka AG. Fresh stock solutions of heme (0.1 mg/ml) were made by adding the hemin to 50% ethanol, followed by a careful dropwise addition with stirring of 1 M NaOH until the entire hemin dissolved and subsequently diluted in the buffer R2 (50 mM Tris–HCl, pH 8.0, 50 mM NaCl, and 10 mM sodium dithionite). This solution was then passed through a 0.2 μm filter into Eppendorf tube. Concentrations of the heme were determined spectrophotometrically, using an extinction coefficient of 385=56 mM−1 cm−1 in buffer R2 [25]. The refolding experiments were carried out in buffer R1 (50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 0.3% SDS, 10 mM sodium dithionite and 7 M urea). The soluble MBP–cytochrome b6 was prepared by adding a 1.5-fold molar excess of the heme to MBP–apocytochrome b6 in buffer R1. After incubating for 30 min at room temperature, the mixture was dialysed in a few steps against the buffer R1, containing a decreased concentration of urea (from 7 to 0 M). The folding mixture was centrifuged at 6000×g for 15 min to remove precipitated protein, loaded on a Sephadex G-25: PD 10 column (Pharmacia), and concentrated using a Centricon-30 filter (Amicon) to remove free heme. |
| Refolding Assay | Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |