Refolding Record:
| Protein | |
|---|---|
| Protein Name | Vascular endothelial growth factor |
| Abbreviated Name | VEGF |
| SCOP Family | Platelet-derived growth factor-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P15692 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | VEGF-165, splice isoform |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 165 |
| Molecular Weight | 19016.0 |
| Pi | 5.85 |
| Molecular Weight | 19016.0 |
| Disulphides | 3 |
| Full Sequence |
APMA EGGGQNHHEV VKFMDVYQRS YCHPIETLVD IFQEYPDEIE YIFKPSCVPL MRCGGCCNDE GLECVPTEES NITMQIMRIK PHQGQHIGEM SFLQHNKCEC RPKKDRARQE KKSVRGKGKG QKRKRKKSRY KSWSVYVGAR CCLMPWSLPG PHPCGPCSER R
|
| Notes | 3 intrachain disulfide bonds, 2 interchain disulfide bonds between dimer subunits |
| Expression | |
|---|---|
| Report | Christinger HW, Muller YA, Berleau LT, Keyt BA, Cunningham BC, Ferrara N, de Vos AM. (1996) Proteins: Structure, Function, and Genetics, 26, 353-357 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | not stated |
| Expression Protocol | not stated |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20mM Tris pH 7.5, 5mM EDTA |
| Solubilization Buffer | 7.5M urea, 20mM Tris pH 7.5 |
| Refolding Buffer | 20mM TrisHCl pH 8.4, 0.4M sodium chloride, 1mM cysteine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.75mg/ml |
| Refolding Time | 24h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Cells were homogenized in 20mM Tris pH 7.5, 5mM EDTA and passed through a French press. The homogenate was centrifuged (15min, 4000g), then the inclusion bodies were resuspended and re-centrifuged. The pellet was dissolved in solubilization buffer and stirred for 1h in 20mM DTT. The protein was then diluted to 0.75mg/ml and dialysed for 24h against refolding buffer. The protein was then purified by ion exchange chromatography (S-Sepharose), a copper chelating columne and a second ion exchange chromatography (SB-Toyo Pearl) |
| Refolding Assay | Crystallography,SDS-PAGE,Receptor binding,Mass spectrometry |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | .3-.4mg/g cell |
| Purity | n/a |
| Notes | Refolding performed with and without 7microM CuCl2 in refolding buffer, no difference in yield observed |