Refolding Record:
| Protein | |
|---|---|
| Protein Name | Manganese-binding protein |
| Abbreviated Name | MntC |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Synechochystis sp. PCC 6803 |
| UniProt Accession | Q79EF9 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | no lipid-anchoring domain |
| Chimera | the first 25aa. of MntC replaced by 16aa. of pET-3XC vector |
| Variants | n/a |
| Chain Length | 305 |
| Molecular Weight | 33591.6 |
| Pi | 4.36 |
| Molecular Weight | 33591.6 |
| Disulphides | 1 |
| Full Sequence |
GTAEV TTSNAPSEEV TAVTTEVQGE TEEKKKVLTT FTVLADMVQN VAGDKLVVES ITRIGAEIHG YEPTPSDIVK AQDADLILYN GMNLERWFEQ FLGNVKDVPS VVLTEGIEPI PIADGPYTDK PNPHAWMSPR NALVYVENIR QAFVELDPDN AKYYNANAAV YSEQLKAIDR QLGADLEQVP ANQRFLVSCE GAFSYLARDY GMEEIYMWPI NAEQQFTPKQ VQTVIEEVKT NNVPTIFCES TVSDKGQKQV AQATGARFGG NLYVDSLSTE EGPVPTFLDL LEYDARVITN GLLAGTNAQQ
|
| Notes | Protein cloned, expressed and purified without the lipid-anchoring domain - the first 25 a.a. of MntC were replaced by 16 a.a. of pET-3XC vector, could not find sequence for pET-3XC). Chain length, MW, pI calculations based on deducible sequence: |
| Expression | |
|---|---|
| Report | Adir N, Rukhman V, Brumshtein B, Anati R (2002) Acta Crystallographica Section D, 58, 1476-1478 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET-3XC |
| Expression Protocol | Cells were grown at 37degC in LB broth supllemented with 100mg/ml ampicillin. Protein expression was induced at mid-logarithmic phase by the addition of 1mM IPTG and growth for a further 4h. Cells were harvested by centrifugation then resuspended in 100mM NaCl, 10mM TrisHCl pH 8.0, 1mM EDTA. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M Urea |
| Refolding Buffer | 20mM TrisHCl pH 8.0, 20mM Mn |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were disrupted by high pressure press treatment at 2MPa and centrifuged (12000rpm, 10min, 4degC). The inclusion bodies were solubilized in 8M urea and then protein refolding was performed by fast dilution into refolding buffer. The refolded protein was purified by ammonium sulfate precipation followed by anion-exchange HPLC. The protein was eluted with a linear gradient of 10-300mM NaCl in 50mM Tris pH 8.0. Selected fractions were pooled and dialysed against 20mM TrisHCl and concentrated. |
| Refolding Assay | Crystallography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |