Refolding Record:
| Protein | |
|---|---|
| Protein Name | Early protein GP1 |
| Abbreviated Name | GP1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bacteriophage phi-29 |
| UniProt Accession | P03679 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 92 |
| Molecular Weight | 10715.3 |
| Pi | 8.04467 |
| Molecular Weight | 10715.3 |
| Disulphides | 0 |
| Full Sequence |
MGKIFDQEKR LEGTWKNSKW GNQGIIAPVD GDLKMIDLEL EKKMTKLEHE NKLMKNALYE LSRMENNDYA TWVIKVLFGG APHGAK HHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Takeuchi A, Makino O, Hirokawa H (1998) Biochemical and Biophysical Research Com, 243, 566-571 |
| Project Aim | Functional Studies |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109(DE3) |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pGEM3Zf+ |
| Expression Protocol | Cells were grown and induced with IPTG, then harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM sodium phosphate pH 7.6, 5M GdnHCl, 10mM imidazole |
| Refolding Buffer | 20mM sodium phosphate pH 7.6, 1M NaCl, 20% glycerol, 1% CHAPS, 10mM imidazole |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.6 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pelleted cells were suspended in 20mM sodium phosphate pH 7.6, 0.5M NaCl, 10mM imidazole. The suspension was then sonicated and centrifuged (12000g, 20min). The insoluble protein was then solubilized in solubilization buffer, centrifuged and filtered. The solubilized protein was loaded onto a Ni column. The protein was then renatured using a linear gradient of urea (6M-1M) in refolding buffer. After renaturation, the protein was eluted in refolding buffer containing 0.5M imidazole. The eluted protein was then buffer exchanged into 20mM TrisHCl pH 8.0, 1% CHAPS using a PD10 column. |
| Refolding Assay | RNA binding |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | |
| Purity | |
| Notes | |