Refolding Record:
| Protein | |
|---|---|
| Protein Name | Putative uncharacterized protein |
| Abbreviated Name | Ehp |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Staphylococcus aureus |
| UniProt Accession | Q99UV2 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 109 |
| Molecular Weight | 12562.7 |
| Pi | 10.4 |
| Molecular Weight | 12562.7 |
| Disulphides | 0 |
| Full Sequence |
MKKNFIGKSILSIAAISLTVSTFAGESHAQTKNVEAAKKYDQYQTNFKKQVNKKVVDAQKAVNLFKRTRTVATHRKAQRAVNLIHFQHSYEKKKLQRQIDLVLKYNTLK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Geisbrecht BV, Bouyain S, Pop M. (2006) Protein Expression and Purification, 46, 23-32 |
| 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 | B834(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 18 h |
| Expression Vector | pT7HMT |
| Expression Protocol | All proteins were expressed from the E. coli strain B834(DE3) and routine manipulations of bacterial cultures were carried out as previously described [3]. During the course of these studies, expression cultures were grown at 37 °C in 250 ml Terrific Broth (TB) and induced at an OD600 of 2.0 by adding IPTG to 1 mM final concentration. The cultures were incubated with vigorous shaking for an additional 18 h to achieve maximal cell density and the induced cells were harvested by centrifugation [3]. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 2.0 = 600 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM sodium phosphate (pH 6.0), 0.5 M NaCl, 10 mM imidazole, 8 M urea |
| Solubilization Buffer | 20 mM sodium phosphate (pH 6.0), 0.5 M NaCl, 0.2 M imidazole, 8 M urea |
| Refolding Buffer | 20 mM Tris (pH 8.0), 0.5 M NaCl |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 5-10 min |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Pellets of induced cells were concomitantly resuspended and lysed by stirring in 5% of the original culture volume of denaturing lysis buffer (0.1 M Tris (pH 8.0), 6 M guanidine–HCl). The cell pellet was stirred at 300–500 rpm for 30 min at room temperature and the solubilized, denatured proteins were separated from the cell debris by centrifugation (30 min at 25,000g). Following centrifugation, the clarified cell extract was decanted and applied by gravity flow to a 2 ml column of Ni2+–NTA Sepharose (Qiagen) that had previously been equilibrated to room temperature in denaturing wash buffer (20 mM sodium phosphate (pH 6.0), 0.5 M NaCl, 10 mM imidazole, 8 M urea). Unbound proteins and contaminants were removed from the column by applying 5 CV of denaturing wash buffer. Tagged proteins were eluted from the resin with 2.5 CV of denaturing elution buffer (20 mM sodium phosphate (pH 6.0), 0.5 M NaCl, 0.2 M imidazole, 8 M urea), though the first 0.5 CV of eluate was discarded since it contained negligible protein.the denatured sample was drawn into an appropriate syringe with needle and refolded by quickly injecting the entire amount into a 10-fold volume excess of rapidly stirring, room temperature native buffer (20 mM Tris (pH 8.0), 0.5 M NaCl). The diluted sample was allowed to stir for 5–10 min longer under these conditions, at which time it was reapplied to the previous 2 ml column of Ni2+–NTA Sepharose that had since been regenerated according to manufacturer’s suggestions and equilibrated in native wash buffer (20 mM Tris (pH 8.0), 0.5 M NaCl, 10 mM imidazole). Excess urea was removed by washing with 5 CV of native wash buffer, and the bound, refolded proteins were eluted as before with 2.5 CV of native elution buffer (20 mM Tris (pH 8.0), 0.5 M NaCl, 500 mM imidazole). |
| Refolding Assay | Analytical gel filtraion chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 10.7 mg/l |
| Purity | n/a |
| Notes | n/a |