Refolding Record:
| Protein | |
|---|---|
| Protein Name | Platelet-derived growth factor |
| Abbreviated Name | PDGF |
| SCOP Family | Platelet-derived growth factor-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P04085 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 127 |
| Molecular Weight | 14305.6 |
| Pi | 9.57421 |
| Molecular Weight | 14305.6 |
| Disulphides | 10 |
| Full Sequence |
SIEEAVPAVCKTRTVIYEIPRSQVDPTSANFLIW
PPCVEVKRCTGCCNTSSVKCQPSRVHHRSVKVAKVEYVRKKPKLKEVQVRLEEHLECACA
TTSLNPDYREEDTGRPRESGKKRKRKRLKPT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Muller C, Rinas U (1999) Journal of Chromatography A, 855, 203-213 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | TG1 |
| Expression Temp | 37.0 |
| Expression Time | 8h |
| Expression Vector | pBS |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.02M TrisHCl pH 8.5, 0.5mM EDTA, 2% (v/v) Triton X-100 |
| Solubilization Buffer | 6M GdnHCl, 0.1M TrisHCl pH 8.5, 0.1M DTT, 1mM EDTA |
| Refolding Buffer | 0.1M TrisHCl pH 7.8, 0.2M GdnHCl, 10mM GSH, 0.25mM GSSG |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | no tag |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Expression was induced at 50g/L cell dry mass by a temperature shift from 30 to 42degC. After 8h incubation, the broth was transferred directly to a high pressure homogenizer. The homogenate was then diluted 4-fold with 0.05M sodium phosphate pH 7.0 and then passed through a continuously working centrifuge at a flow rate of 75L/h. The insoluble cell fraction was washed three times with wash buffer each time followed by centrifugation (8500g, 4degC, 40min). The last centrifugation step was performed with 25ml aliquots at 10000g, 4deg, 40min. The inclusion body pellet was stored at -70degC. After thawing, the inclusion bodies from the 25ml aliquots were resuspended in 5ml solubilization buffer. The suspension was sonicated for 5 min then incubated overnight at room temperature. The solution was centrifuged (26000g, 4degC, 45min). The protein was refolded by dilution into refolding buffer at 25degC. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 45% |
| Purity | |
| Notes | Expression vector carrying a bicistronic operon allowed co-expression of A and B subunits. See same paper for more successful refolding of the same protein by size-exclusion chromatography. |