Refolding Record:
| Protein | |
|---|---|
| Protein Name | Glutamate mutase |
| Abbreviated Name | MutES |
| SCOP Family | Cobalamin (vitamin B12)-binding domain |
| Structure Notes | |
| Organism | Clostridium tetanomorphum |
| UniProt Accession | Q05509 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Heterodimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 616 |
| Molecular Weight | 67738.6 |
| Pi | 5.46368 |
| Molecular Weight | 67738.6 |
| Disulphides | 0 |
| Full Sequence |
MELKNKKWTD EEFFKQREEV LKQWPTGKEV DLQEAVDYLK KVPTEKNFAD KLVRAKEAGI TLAQPRAGVA LLDEHINLLR YLQDEGGADL LPSTIDAYTR QNRYEECEIG IKESEKAGRS LLNGFPGVNH GVKGCRKVLE SVNLPLQARH GTPDSRLLAE IIHAGGWTSN EGGGISYNIP YAKSVPIDKC LKDWQYCDRL
VGFYEEQGVH INREPFGPLT GTLVPPSMSN AVGITEALLA AEQGVKNITV GYGECGNMLQ DIAALRCLEE QTNEYLKAYG YNDVFVTTVF HQWMGGFPQD ESKAFGVIVT ATTIASLAGA TKVIVKTPHE AIGIPTKEAN ASGIKATKMA LNMLEGQRMP MSKELETEMA IIKAETKCIL DKMFELGKGD LAVGTVKAFE TGVMDIPFGP SKYNAGKMMP VRDNLGCVRY LEFGNVPFTE ELKNYNRERL AERAKFEGRE VSFQMVIDDI FAVGKGRLIG RPEK IVLGV IGSDCHAVGN KILDHSFTNA GFNVVNIGVL SSQEDFINAA IETKADLICV SSLYGQGEID CKGLREKCDE AGLKGIKLFV GGNIVVGKQN WPDVEQRFKA MGFDRVYPPG TSPETTIADM KEVLGVE
|
| Notes | Fusion of E and S subunits. UniProt of S subunit: MAMA_CLOTT |
| Expression | |
|---|---|
| Report | Holloway DE, Harding SE, Marsh ENG (1996) Biochem J., 320, 825-830 |
| Project Aim | Structure-Function,Biophysical Studies,Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | TG1recO |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pUC119 |
| Expression Protocol | Cells were grown at 37degC in 2TY culture until OD600 reached 1.0-1.5. Expression was induced by addition of 200mg/L IPTG and the culture was grown for a further 3h |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 1.0-1.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50mM potassium phosphate pH 6.9, 100mM NaCl, 1mM DTT, 1mM EDTA, 0.1mM PMSF, 0.5% Triton X-100 |
| Solubilization Buffer | 0.1M potassium phosphate pH 6.9, 6M GdnHCl, 10mM DTT, 1mM EDTA |
| Refolding Buffer | 0.1M potassium phosphate pH 7.6, 10% glycerol, 1mM DTT |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.6 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 50microg/l |
| Refolding Time | overnight |
| Redox Agent | DTT |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | 12g (wet weight) of cells were resuspended in 30ml wash buffer without Triton X-100. The cells were ruptured by sonication, then centrifuged (15min, 12000g). The pellet was washed (and centrifuged) twice in 25ml wash buffer. The washed inclusion bodies were dissolved in 25ml solubilization buffer and stirred at room temperature for 2h. The solution was then centrifuged (25000g, 15min). The supernatant was then diluted drop-wise to 100ml refolding buffer containing 1M GdnHCl, with gentle stirring, with a final protein concentration of approximately 50microg/ml. The solution was stirred a further 20min and then dialysed overnight against 4L refolding buffer. The dialysate was then cleared by centrifugation (25000g, 15min) and the protein was concentrated |
| Refolding Assay | Enzyme activity,Analytical centrifugation |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | 30% |
| Purity | n/a |
| Notes | n/a |