Refolding Record:
| Protein | |
|---|---|
| Protein Name | 5,10-methylenetetrahydroforate reductase |
| Abbreviated Name | METF |
| SCOP Family | Methylenetetrahydrofolate reductase |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P0AEZ1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Tetramer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 296 |
| Molecular Weight | 33102.7 |
| Pi | 6.0 |
| Molecular Weight | 33102.7 |
| Disulphides | Unknown |
| Full Sequence |
MSFFHASQRDALNQSLAEVQGQINVSFEFFPPRTSEMEQTLWNSIDRLSSLKPKFVSVTYGANSGERDRTHSIIKGIKDRTGLEAAPHLTCIDATPDELRTIARDYWNNGIRHIVALRGDLPPGSGKPEMYASDLVTLLKEVADFDISVAAYPEVHPEAKSAQADLLNLKRKVDAGANRAITQFFFDVESYLRFRDRCVSAGIDVEIIPGILPVSNFKQAKKFADMTNVRIPAWMAQMFDGLDDDAETRKLVGANIAMDMVKILSREGVKDFHFYTLNRAEMSYAICHTLGVRPGL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Okuda H, Sakuhana C, Yamamoto R, Kawai R, Mizukami Y, Matsuda K. (2007) Biometals, 20, 903-910 |
| Project Aim | Folding |
| Fusion | None |
| 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 | 12 h |
| Expression Vector | pET22b |
| Expression Protocol | Recombinant METF protein fused with additional amino residues (LEHHHHHH) from pET22b (+) vector at the C terminus was expressed in E. coli strain BL21 (DE3) and extracted from the cells by the same procedure as for GroEL with buffer D (50 mM Tris–HCl, pH 7.6, 500 mM NaCl and 5 mM imidazole). The crude extract was purified by Ni-NTA affinity resin (Novagen) followed by gel filtration using a Superdex 200 GL column with refolding buffer. The purity of the proteins was checked by SDS and native PAGE as described for GroEL. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris–HCl, pH 7.6, 500 mM NaCl and 5 mM imidazole |
| Solubilization Buffer | 4 M Gdn–HCl for 1 h at 25°C |
| Refolding Buffer | 0.5 μM GroEL, 1 μM GroES, 2 mM ATP-Na3, 10 mM divalent cation, 1 mg/ml BSA and 0.5 μM FAD |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 37.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | METF refolding assay Unless otherwise stated, the molar concentrations of METF, GroEL and GroES are expressed as those of 4, 14 and 7 mer, respectively. METF (10 μM) was denatured with 4 M Gdn–HCl for 1 h at 25°C. Denatured METF was diluted 100-fold at 37°C into refolding buffer containing 0.5 μM GroEL, 1 μM GroES, 2 mM ATP-Na3, 10 mM divalent cation, 1 mg/ml BSA and 0.5 μM FAD. The refolding reaction was stopped by the addition of 40 mM CDTA. METF activity was assayed at 25°C for 15 min by measuring absorbance at 340 nm in METF assay solution (50 mM Tris–HCl, pH 7.2, 200 μM NADH, 180 μM menadione, 2 mM EDTA and 1 mg/ml BSA). The refolding yield was determined as the percentage of activity of the refolding enzyme relative to that of the native enzyme. |
| Refolding Assay | ATP hydrolysis |
| Refolding Chaperones | GroEL,GroES |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |