Refolding Record:
| Protein | |
|---|---|
| Protein Name | p55-hGRF (truncated form of Ras-GDP exchange factor) |
| Abbreviated Name | p55-hGRF |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q13972 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | shorter construct p39-hGRF - C-terminal 339 residues of the protein |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 339 |
| Molecular Weight | 39366.1 |
| Pi | 5.93771 |
| Molecular Weight | 39366.1 |
| Disulphides | 0 |
| Full Sequence |
PDQRNGDKEF VIRRAATNRV LNVLRHWVSK HSQDFETNDE LKCKVIGFLE EVMHDPELLT QERKAAANII RTLTQEDPGD NQITLEEITQ MAEGVKAEPF ENHSALEIAE QLTLLDHLVF KKIPYEEFFG QGWMKLEKNE RTPYIMKTTK HFNDISNLIA SEIIRNEDIN ARVSAIEKWV AVADICRCLH NYNAVLEITS SMNRSAIFRL KKTWLKVSKQ TKALIDKLQK LVSSEGRFKN
LREALKNCDP PCVPYLGMYL TDLAFIEEGT PNYTEDGLVN FSKMRMISHI IREIRQFQQT AYKIEHQAKV TQYLLDQSFV MDEESLYESS LRIEPKLPT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Meyer P, Janin J, Baudet-Nessler S (1999) Eur J Biochem., 263, 806-816 |
| Project Aim | Structure-Function |
| 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 | overnight |
| Expression Vector | pET3d |
| Expression Protocol | 1L of LB broth containing 100microg/ml ampicillin was inoculated with 10ml overnight culture. Cells were incubated until A600 reached 1.0, expression was induced with 1mM IPTG and incubation continued overnight. Cells were harvested by centrifugation (7000g, 4degC, 10min) and the pellet was stored at -80degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 1.0 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 2.0M GdnHCl |
| Solubilization Buffer | 6.0M GdnHCl, 40mM TrisHCl pH 7.0, 500mM NaCl, 1mM EDTA, 5mM DTT |
| Refolding Buffer | 50mM TrisHCl pH 7.0, 500mM NaCl, 1mM EDTA, 8-12% glycerol, 100microM Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 36h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cell pellet was resuspended to 0.3g/ml in 50mM TrisHCl pH 8.0, 100mM NaCl, 1mM EDTA, 2mM DTT, 250microM PMSF and sonicated (6x30sec) on ice. The lysate was centrifuged (25000g, 15min) and the pellet was washed twice in wash buffer. The pellet was then dissolved in solubilization buffer and stirred on ice for 30min. Refolding was performed by a 2-step dialysis process. The solubilized protein was centrifuged (25000g, 4degC, 30min) and the supernatant was diluted to 0.35mg/ml in solubilization buffer. The protein was then dialyzed against 10 volumes of refolding buffer without glycerol containing 4.0M urea at 4degC for one day. The dialysis was then continued for 2 days at 4degC against 50 volumes of refolding buffer. The dialyzed solution was then centrifuged (20min, 20000g, 4degC) to remove precipitate |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 8-12% |
| Refolding Yield | 100mg/L culture |
| Purity | |
| Notes | |