Refolding Record:
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 3 |
| Molecular Weight | 203.2 |
| Pi | 5.52 |
| Molecular Weight | 203.2 |
| Disulphides | Unknown |
| Full Sequence |
n/a
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bajorunaite E, Sereikaite J, Bumelis VA. (2007) Protein journal, 26, 547-555 |
| Project Aim | Protein refolding |
| 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 | 200 min |
| Expression Vector | pET21a |
| Expression Protocol | Escherichia coli strain BL21(DE3) harboring the plasmid pET21a+/pGH or pET21a+/mGH was cultivated in a batch fermentation process as previously described [21, 22]. The expression of target protein was induced with 1 mM isopropyl β-d-thiogalactoside. Both recombinant pGH and mGH were expressed as insoluble proteins and accumulated as inclusion bodies.About 4 g of frozen biomass was homogenized in 40 mL of 0.1 M Tris–HCl buffer pH 7.2, containing 5 mM EDTA and the cells were sonicated 5 min on ice using a Sonics Vibracell VCX 750. PMSF to a final concentration of 1 mM was added just before the sonication. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 2 M urea, 1 M NaCl, 5 mM EDTA, 1 mM PMSF in 0.1 M Tris–HCl buffer pH 8.0 |
| Solubilization Buffer | 40 mL of 0.1 M Tris–HCl buffer pH 8.0 containing 8 M urea and 20 mM reduced glutathione |
| Refolding Buffer | 0.1 M Tris–HCl buffer pH 8.0 containing 6 mM oxidized glutathione in order to reduce urea concentration to 3 M , 1 M Arginine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 20 h |
| Redox Agent | GSSG |
| Redox Agent Concentration | 20 mM,6 mM |
| Refolding Protocol | Refolding and Purification of pGH The solution of solubilized pGH or mGH in 0.1 M Tris–HCl buffer pH 8.0 containing 8 M urea and 20 mM reduced glutathione (from Sect. 2.3) was diluted with 0.1 M Tris–HCl buffer pH 8.0 containing 6 mM oxidized glutathione in order to reduce urea concentration to 3 M. Arginine was added to the dilution buffer to a final concentration of 1 M in the renaturation mixture. Since arginine–HCl was used prior to adding oxidized glutathione pH of dilution solution was adjusted to pH 8.0. The renaturation reaction was carried out for 20 h at 4 °C with gentle stirring. Finally, the solution was centrifuged at 13,000 g for 25 min at 4 °C and chaotropic agent, thiol-compounds and arginine were removed by size exclusion chromatography using a Sephadex G-25 gel filtration column (2.5 × 80.5 cm), equilibrated with 0.025 M Tris–HCl buffer pH 8.0 at a flow rate of 1.5 mL/min. Fractions containing pGH or mGH were pooled and loaded on Q-Sepharose column (2.5 × 12 cm) previously equilibrated with 0.025 M Tris–HCl buffer pH 8.0 at a flow rate of 2 mL/min. Unbound proteins were washed out with the same buffer, and bound proteins were eluted with a linear salt gradient as follows: 1 bed volume of 0 M NaCl to 0.15 M NaCl, 2 bed volumes of 0.15 M NaCl to 0.3 M NaCl and 2 bed volumes of 0.3 M NaCl to 1 M NaCl in 0.025 M Tris–HCl buffer pH 8.0. The peak which appeared in the range of 0.3–1 M of NaCl and contained pGH or mGH was pooled. The salt concentration was adjusted approximately to 2 M, and the protein solution was applied on Phenyl-Sepharose column (1.5 × 13.5 cm) previously equilibrated with 0.025 M Tris–HCl buffer pH 8.0 containing 2 M NaCl at a flow rate of 1 mL/min. Unbound proteins were washed out with the same buffer, and bound proteins were eluted with 10 bed volumes of linear NaCl gradient (0.8–0.2 M) in 0.025 M Tris–HCl buffer pH 8.0. Fractions containing pGH or mGH were pooled and stored at −20 °C. |
| Refolding Assay | SDS-PAGE,RP-HPLC |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 3 mM |
| Refolding Yield | 50-60% |
| Purity | 88 ± 3 |
| Notes | n/a |