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 | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 53 |
| Molecular Weight | 6222.0 |
| Pi | 4.77848 |
| Molecular Weight | 6222.0 |
| Disulphides | 3 |
| Full Sequence |
NSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lee JY, Yoon CS, Chung IY, Lee YS, Lee EK (2000) Biotechnol. Appl. Biochem., 31, 245-248 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15[rep4] |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pQE30 |
| Expression Protocol | In a shake-flask culture, RM medium (40 g/l glucose, 20 g/l casamino acid, 6 g/l Na2HPO4, 3 g/l KH2PO4, 1 g/l NH4Cl, 0.5 g/l NaCl, 0.095 g/l MgCl2, 0.1 g/l ampicillin and 0.05 g/l kanamycin) was used as suggested by Invitrogen, except that glucose was replaced with glycerol. In a 5-l jar fermentation, the same medium was used at pH 7, 37deg C, 400 revs./min and at an aeration rate of 1 vol. of air/vol. of medium/min. EGF expression was induced by the addition of 2 mM IPTG. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Detergent precipitation/sequestration |
| Wash Buffer | 10mM EDTA, 0.2M Na2HPO4, pH 7.5 |
| Solubilization Buffer | 1.0% (w/v) SDS, 46mM Na2CO3, pH 9.8 |
| Refolding Buffer | 46mM Na2CO3, 5mM L-cysteine, 5mM L-cystine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 9.8 |
| Refolding Temperature | 22.0 |
| Protein Concentration | |
| Refolding Time | 14-16h |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | 5mM/5mM |
| Refolding Protocol | Cells were harvested by centrifugation at 6600 g and washed in wash buffer A (20 mM EDTA/0.1 M Na2HPO4, pH 7.8). The washed cells were disrupted by ultrasonic disintegrator (model 550, Fisher Scientific) operated for 10 cycles at 7.5 intensity. EGF IB pellets were obtained by centrifuging the cell lysate at 5600 g for 30 min, and then washed by wash buffer B (10 mM EDTA/0.2 M Na2HPO4, pH 7.5). SDS was used to dissolve the IB as described previously by Kim et al. [7]. An aliquot of the washed IB (0.66 g dry weight) was added in 100 ml of dissolution buffer [1.0% (w/v) SDS in 46 mM Na2CO3, pH 9.8] at about 22 °C for 24 h. Rapid cooling to 4 °C precipitated approx. two-thirds of the SDS, and the precipitate was removed by centrifugation. The residual SDS in the supernatant was removed by Amberlite IRA402 (Rohm and Haas, Philadelphia, PA, U.S.A.) anion-exchange chromatography. To the eluate 5 mM L-cysteine was added for reduction. After 1 h, 5 mM L-cystine was added for oxidative refolding for 14?6 h at about 22 °C. Refolding aggregate was ultrafiltered by 100-kDa molecular-mass cut-off membrane, and the permeate was fed to Ni2+-nitrilotriacetate (Ni2+-NTA)-agarose affinity chromatography (Qiagen). The bound EGF was eluted with the carbonate buffer containing 250 mM imidazole (Sigma). The EGF fractions were dialysed and then subjected to the second refolding using the same procedure as in the first refolding. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 49.6% |
| Purity | |
| Notes | |