Refolding Record:
| Protein | |
|---|---|
| Protein Name | Transcription factor Sp1 |
| Abbreviated Name | Sp1 |
| SCOP Family | Classic zinc finger |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P08047 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | DNA binding domain aa.612-778 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 167 |
| Molecular Weight | 18164.8 |
| Pi | 9.67825 |
| Molecular Weight | 18164.8 |
| Disulphides | 1 |
| Full Sequence |
SEGRGSGDP GKKKQHICHI QGCGKVYGKT SHLRAHLRWH
TGERPFMCTW SYCGKRFTRS DELQRHKRTH TGEKKFACPE CPKRFMRSDH LSKHIKTHQN KKGGPGVALS VGTLPLDSGA GSEGSGTATP SALITTNMVA MEAICPEGIA RLANSGINVM QVADLQSI
|
| Notes | DNA binding domain comprises 3 zinc fingers |
| Expression | |
|---|---|
| Report | Kuwahara J and Coleman JE (1990) Biochemistry, 29, 8627-8631 |
| Project Aim | Structural Studies |
| 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 | 4h |
| Expression Vector | pAR3039 |
| Expression Protocol | Cells were grown until A600 reached 1, expression was induced with IPTG and cells were harvested 4h later. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 1 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | N/A |
| Solubilization Buffer | 5M urea, 10mM TrisHCl, 50mM NaCl, 1mM beta-mercaptoethanol pH 7.5 |
| Refolding Buffer | 10mM TrisHCl, 0.2M NaCl, 1mM beta-mercaptoethanol, 50microM ZnSO4, 5% glycerol pH 7.5 |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 96h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Cells from 2.4L broth (10g wet weight) were resuspended in 20ml of 10mM TrisHCl, 1mM beta-mercaptoethanol, 1mM EDTA, 50mM NaCl, pH 7.5 and lysed with lysozyme and deoxycholate (0.04%) followed by DNaseI (50microg/ml) treatment. Following centrifugation, the pellet was resuspended in solubilization buffer and loaded onto a Trisacryl SP column preequilibrated with the same buffer at 25degC. The protein was eluted with a 50mM to 1M gradient of NaCl, 25degC. The protein was refolded by dialysis against refolding buffer. After two changes of dialyzate, the protein was dialyzed for four changes of 24h each against the refolding buffer with no metals. |
| Refolding Assay | Ultraviolet (UV) Absorbance |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | |
| Purity | |
| Notes | |