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 | Heterodimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 285 |
| Molecular Weight | 32393.8 |
| Pi | 10.0297 |
| Molecular Weight | 32393.8 |
| Disulphides | 8 |
| Full Sequence |
SIEE AVPAVCKTRT VIYEIPRSQV DPTSANFLIW PPCVEVKRCT GCCNTSSVKC QPSRVHHRSV KVAKVEYVRK KPKLKEVQVR LEEHLECACA TTSLNPDYRE EDTGRPRESG KKRKRKRLKP T SLGSLTIAE PAMIAECKTR TEVFEISRRL IDRTNANFLV WPPCVEVQRC SGCCNNRNVQ CRPTQVQLRP VQVRKIEIVR KKPIFKKATV TLEDHLACKC ETVAAARPVT RSPGGSQEQR
AKTPQTRVTI RTVRVRRPPK GKHRKFKHTH DKTALKETLG A
|
| Notes | UniProt for B chain: PDGFB_HUMAN |
| 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 | Expression was induced at 50g/L cell dry mass by a temperature shift from 30 to 42degC and incubated for 8h. |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| 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 | A: 0.1M TrisHCl pH 7.8, 4M GdnHCl, B: 0.1M TrisHCl pH 7.5, 10mM GSH, 0.25mM GSSG |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 10mM/0.25mM |
| Refolding Protocol | 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 then passed down a size exclusion column equilibrated with a mixture of 12% refolding buffer A and 88% refolding buffer B. Samples were loaded with 10ml of 2M GdnHCl, elution conditions were later changed at 0.5M GdnHCl (Refolding buffer B). |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 75% |
| Purity | |
| Notes | Expression vector carrying a bicistronic operon allowed co-expression of A and B subunits. See same paper for less successful refolding of the same protein by dilution |