Refolding Record:
| Protein | |
|---|---|
| Protein Name | GATA-type transcription factor |
| Abbreviated Name | MED-1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | C. elegans |
| UniProt Accession | Q9GSP3 |
| 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 | 194 |
| Molecular Weight | 22075.0 |
| Pi | 8.95 |
| Molecular Weight | 22075.0 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSH MAYPYPVFNAENVFDNTQQQVGFYDYSTPFNGTYSFTTDYSYYNNYYDYVNTYASYYPTA
MDSSSLNISSTTGSPNSSHFTTFTHFSTPSTSPSTSTQSSTTPSNSDNKKSFQCSNCSVT
ETIRWRNIRSKEGIQCNACFIYQRKYNKTRPVTAVNKYQKRKLKVQETNGVDSF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Broitman-Maduro, G., Maduro, M. F., Rothman, J. H. (2005) Dev Cell, 8, 427-433 |
| Project Aim | 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 | CodonPlus-RIL |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pEt-15b |
| Expression Protocol | Induced cells were allowed to grow overnight before they were harvested by centrifugation, and resuspended in BugBuster(Novagen) for cell lysis. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.3 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Chemicals |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M Urea, 50mM Tris, pH 7.5 and 25mM DTT on ice |
| Refolding Buffer | 1M NDSB-256, Novagen, 50mM Tris, pH 7.5, 500mM NaCl, 0.5mM DTT, 0.5mM zinc sulfate, 10% glycerol |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 37.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.5 |
| Refolding Protocol | Inclusion bodies were washed 2x with Bugbuster, then 2x with water before solubilization. They were then renatured by 10x dilution with refolding buffer. The refolded extract was then Ni2+ affinty column before the tag was removed by thrombin cleavage. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |