Refolding Record:
| Protein | |
|---|---|
| Protein Name | Dimethylglycine dehydrogenase |
| Abbreviated Name | Dimethylglycine dehydroge |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | Q63342 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 846 |
| Molecular Weight | 95029.2 |
| Pi | 6.57 |
| Molecular Weight | 95029.2 |
| Disulphides | 0 |
| Full Sequence |
MSPIHHHHHHLVPRGSEASNSTMSPPEWKDRAETVIIGGGCVGVSLAYHLAKAGMRDVVLLEKSELTAGSTWHAAGLTTYFHPGINLKKIHYDSIKLYERLEEETGQVVGFHQPGSIRLATTPERVDEFKYQMTRTNWHATEQYIIEPEKIHELFPLLNMDKILAGLYNPGDGHIDPYSLTMALATGARKYGVLLKYPAPVTSLKPRPDGTWDVETPQGSVRANRIVNAAGFWAREVGKMIGLDHPLIPVQHQYVVTSTIPEVKALKRELPVLRDLEGSYYLRQERDGLLFGPYESQEKMKLQASWVAHGVPPGFGKELFESDLDRITEHVEAAMEMVPVLKKADIINIVNGPITYSPDILPMVGPHQGVRNYWVAIGFGYGIIHAGGVGKYLSDWILHGEPPFDLIELDPNRYGKWTTTQYTEAKARESYGFNNIVGYPKEERFAGRPTQRVSGLYKILESKCSMGFHAGWEQPHWFYKPGQDTQYRPSFRRTNWFRPVGSEYKQVMQRVGVIDLSPFGKFNIKGQDSTQLLDHLCANVIPKVGFTNISHMLTPRGRVYAELTVSHQSPGEFLLITGSGSELHDLRWIEEAAVRGGYDVEIRNITDELGVLGVAGPYARRVLQKLTSEDLSDDVFKFLQTKSLKISDIPVTAIRISYTGELGWELYHRREDSAALYERIMNAGQEEGIDNFGTYALNALRLEKAFRAWGSEMNCDTNPLEAGLDYFIKLNKPADFTGKQALKQIKAKGLKRRLVCLTLATDDVDPEGNESVWYKGKVIGNTTSGSYSYSIQKSLAFAYVPVELSEVGQQVEVELLGKNYPATIIQEPLVLTEPTRTRLQKDGRKS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Brizio, C., Brandsch, R., Bufano, D., Pochini, L., Indiveri, C., Barile, M (2004) Protein Expression and Purification, 37, 434-442 |
| Project Aim | Folding,Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109 |
| Expression Temp | 30.0 |
| Expression Time | 8h |
| Expression Vector | pH6EX3 |
| Expression Protocol | Overnight culture was used to inoculate 1L LB supplemented with 100microg/ml ampicillin. This culture was induced, an allowed to incubate. Bacteria were harvested by centrifugation(3000g. 10min, 4degC and pellets stored at -20degC. Pellet was thawed on ice for 15min and resuspended in 30ml Buffer A (500mM NaCl, 40mM Hepes-Na, pH 7.4) supplemented with 0.2mL of Protease Inhibitor Cocktail. Cells were disrupted and inclusion bodies isolated by centrifugation (20,000g, 30min, 4degC). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 1.8-3.3% SDS, 100 mM potassium phosphate, pH 7.0. |
| Refolding Buffer | 100 mM potassium phosphate, pH 7.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 30.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were solubilized by the addition of solubilization buffer and were then refolded by 80 fold dilution with refolding buffer. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Using 3.3% SDS had the best results for protein activity after refolding. |