Refolding Record:
| Protein | |
|---|---|
| Protein Name | Secreted protein acidic and rich in cysteine |
| Abbreviated Name | SPARC |
| SCOP Family | Osteonectin |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P09486 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 295 |
| Molecular Weight | 33894.0 |
| Pi | 4.85 |
| Molecular Weight | 33894.0 |
| Disulphides | 7 |
| Full Sequence |
MAPQQEALPDETEVVEETVAEVTEVSVGANPVQVEVGEFDDGAEETEEEVVAENPCQNHHCKHGKVCELDENNTPMCVCQDPTSCPAPIGEFEKVCSNDNKTFDSSCHFFATKCTLEGTKKGHKLHLDYIGPCKYIPPCLDSELTEFPLRMRDWLKNVLVTLYERDEDNNLLTEKQKLRVKKIHENEKRLEAGDHPVELLARDFEKNYNMYIFPVHWQFGQLDQHPIDGYLSHTELAPLRAPLIPMEHCTTRFFETCDLDNDKYIALDEWAGCFGIKQKDIDKDLVILEHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bassuk, J. A., Braun, L. P., Motamed, K., Baneyx, F., Sage, H (1996) Int J Biochem Cell Biol, 28, 1013-1043 |
| Project Aim | Folding |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET-22b |
| Expression Protocol | Cells were grown in 1 L pilot-scale fermentors in Luria Broth that contained 50microg/ml carbenicill |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5-0.7 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | not stated |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea, 0.1 M NaH2PO4, 0.01 M TrisHCl, pH 8.0, 0.05 M NH4OH, 1% octylthioglucoside |
| Refolding Buffer | 0.1 M NaH2PO4, 0.01 M TrisHCl, 0.5 NaCl, 20% glycerol, pH 7.8 supplemented with 4.7 M Urea |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Insoluble proteins were solubilized by the addition of solubilization buffer of which the pH was adjusted to 11.0 with NaOH. This solution was stirred for 2 hrs at ambient temperature before the pH was adjusted with HCl, to 7.5. DTT and DTE were then added to a final concentration of 0.05 M and stirring continued for 1h. The mixture was then centrifuged (20,000g, 45min, 25degC) and preformed to Gel electrophoresis ensure the complete extraction of rSPARC from the insoluble pellet. The supernatant was then dialyzed against refolding buffer supplemented with different volumes of urea at differing time points. 2-4 hrs: 10 vol, 14-18hrs: 70 vol, 1 hr: 10 vol. Purification on a Ni-NTA agarose column was then preformed. |
| Refolding Assay | Immunoassay |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |