Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prostate-specific antigen |
| Abbreviated Name | PSA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07288 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 238 |
| Molecular Weight | 26089.0 |
| Pi | 7.25 |
| Molecular Weight | 26089.0 |
| Disulphides | Unknown |
| Full Sequence |
IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hsieh MC, Cooperman BS. (2000) Biochemica et Biophysica Acta, 1481, 75-87 |
| Project Aim | Expression and charactrization |
| Fusion | None |
| 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 | 3-4 h |
| Expression Vector | pET |
| Expression Protocol | E. coli strain BL21(DE3) containing pET-prorPSA, was grown at 37³C in 15 l of LB medium containing 0.1 mg/ml ampicillin. When A600 reached 0.4-1.0, isopropyl-L-D-thiogalactoside was added at 1 mM final concentration to induce pro-rPSA expression, and growth was continued for another 3^4 h. Bacterial cells were harvested, quickly frozen, and stored at 370C. Inclusion bodies were isolated ac- cording to Nagai and Thogersen [11]. Brie£y, 20 g of cell pellet were suspended in 16 ml of lysis bufer (50 mM Tris^HCl, pH 8.0, 25% sucrose (w/v), 1 mM EDTA). Lysozyme was then added (4 ml, dissolved in lysis bu¡er) to a final concentration of 2 mg/ml. Following incubation for 30 min on ice, MgCl2 (10 mM), MnCl2 (1 mM), and DNase I (10 mg/ml) were added (final concentrations) and in- cubation was continued for another 30 min. Detergent bufer (40 ml of 20 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 1% deoxycholic acid, and 1% IGEPAL CA- 630) was then added and the solution was centrifuged at 5000g for 10 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4-1.0 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea, 50 mM Tris^HCl, pH 8.5, 150 mM NaCl, 0.1 M NH4 Cl, 2 mM EDTA, 10 mM benzamidine, and 0.1 M 2-mercaptoethanol |
| Refolding Buffer | 50 mM Tris^HCl, pH 8.5, 0.5 M arginine, 3 mM reduced glutathione, 0.3 mM oxidized glutathione, 5 mM EDTA, and 0.1% PEG |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 3/0.3 mM |
| Refolding Protocol | The final concentration of protein in refolding buffer (50 mM Tris^HCl, pH 8.5, 0.5 M arginine, 3 mM reduced glutathione, 0.3 mM oxidized glutathione, 5 mM EDTA, and 0.1% PEG) was brought to V10 Wg/ml, requiring a total volume of 8 l. As a practical matter, refolding was carried out on 2 l portions at a time (placing 200 ml in each of ten 500 ml beakers overnight at 4C - partial filling of the beaker increases oxygen availability) over 4 successive days. Each 2 l portion was dialyzed against 30 l of 10 mM Tris-HCl, pH 7.8, with three changes, clarified by centrifugation at 10 000Ug for 10 min, concentrated to V300 ml by ultrafiltration using a PM10 membrane (Amicon), and brought to 1 M ammonium sulfate. The solution was then clarified by centrifugation and applied to a hydrophobic interaction phenyl Sepharose column (25 ml, Pharma cia), pre-equilibrated with 1 M ammonium sulfate in 10 mM Tris^HCl, pH 7.8. A linear ammonium sulfate gradient (400 ml, 1 to 0 M) in 10 mM Tris-HCl bu¡er, pH 7.8, was used to elute aggregates and misfolded pro-rPSA. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |