Refolding Record:
| Protein | |
|---|---|
| Protein Name | Epidermal Growth Factor |
| Abbreviated Name | EGP |
| SCOP Family | EGF-type module |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01133 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | Cell binding domain of Fibronectin-EGF |
| Variants | n/a |
| Chain Length | 332 |
| Molecular Weight | 36602.0 |
| Pi | 5.30506 |
| Molecular Weight | 36602.0 |
| Disulphides | 3 |
| Full Sequence |
P TDLRFTNIGP DTMRVTWAPP PSIDLTNFLV RYSPVKNEED VAELSISPSD NAVVLTNLLP GTEYVVSVSS VYEQHESTPL
RGRQKTGLDS PTGIDFSDIT ANSFTVHWIA PRATITGYRI RHHPEHFSGR PREDRVPHSR NSITLTNLTP GTEYVVSIVA LNGREESPLL IGQQSTVSDV PRDLEVVAAT PTSLLISWDA PAVTVRYYRI TYGETGGNSP VQEFTVPGSK STATISGLKP GVDYTITVYA VTGRGDSPAS SKPISINYRT EIDKPS MA NSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kawase Y, Ohdate Y, Shimojo T, Taguchi Y, Kimizuka F, Kato I (1992) FEBS Letters, 298, 126-128 |
| Project Aim | Drug Studies |
| Fusion | N-terminal fibronectin cell-binding domain |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HB101 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pUC118NT |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM TrisHCl pH 8.0, 10mM EDTA, 100mM NaCl, 0.5% Triton X-100 |
| Solubilization Buffer | 20mM TrisHCl pH 8.0, 250mM DTT, 6M urea |
| Refolding Buffer | 20mM TrisHCl pH 7.5, 1.0mM reduced glutathione, 0.1mM oxidized glutathione |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1.0mM/0.1mM |
| Refolding Protocol | The cell pellet obtained from 2L culture was supended in 50ml 50mM TrisHCl pH 8.0, 25% sucrose, 1mM EDTA and incubated with 4mg lysozyme at 4degC for 30min followed by sonication. 150microL of 1.0M MgCl2 and 7500 units of DNaseI were added and the mixture was incubated at 37degC for 30min. After the addition of 10ml of 20mM TrisHCl pH 7.5, 0.2M NaCl, 1.0% deoxycholate and 1.0% NP-40. The inclusions were washed first with wash buffer, secondly with 20% ethylene glycol and finally with 2.0% NP-40. The inclusion bodies were then dissolved in 25ml solubilization buffer and loaded onto a column of DEAE-Toyopearl 650S (15ml) equilibrated with 20mM TrisHCl pH 8.0 containing 10mM DTT. After the column was washed in the same buffer, the protein was eluted with a stepwise increase in NaCl concentration. Selected fractions were pooled and dialyzed for 40h at 4degC against refolding buffer, then dialyzed against PBS and lyophilized. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |