Refolding Record:
| Protein | |
|---|---|
| Protein Name | Gelonin-acetylcholine receptor alpha sub unit |
| Abbreviated Name | Gelonin-AChR |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Gelonium multiflorum |
| UniProt Accession | P33186-P02710 |
| 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 | 428 |
| Molecular Weight | 48673.7 |
| Pi | 6.45 |
| Molecular Weight | 48673.7 |
| Disulphides | Unknown |
| Full Sequence |
GLDTVSFSTKGATYITYVNFLNELRVKLKPEGNSHGIPLLRKKCDDPGKCFVLVALSNDNGQLAEIAIDVTSVYVVGYQVRNRSYFFKDAPDAAYEGLFKNTIKTRLHFGGSYPSLEGEKAYRETTDLGIEPLRIGIKKLDENAIDNYKPTEIASSLLVVIQMVSEAARFTFIENQIRNNFQQRIRPANNTISLENKWGKLSFQIRTSGANGMFSEAVELERANGKKYYVTAVDQVKPKIALLKFVETRLVANLLENYNKVIRPVEHHTHFVDITVGLQLIQLISVDEVNQIVETNVRLRQQWIDVRLRWNPADYGGIKKIRLPSDDVWLPDLVLYNNADGDFAIVHMTKLLLDYGKIMWTPPAIFKSYCEIIVTHFPFDQQNCTMKLGIWTYDGTKVSISPESDRPDLSTFMESGEEWVIKESLSNN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hossann M, Li Z, Shi Y, Kreilinger U, Büttner J, Vogel PD, Yuan J, Wise JG, Trommer WE. (2006) Protein Expression and Purification, 46, 73-84 |
| Project Aim | Drug Studies |
| Fusion | N-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 | 3 h |
| Expression Vector | pET28a |
| Expression Protocol | The expression of the fusion protein was carried out as described above for gelonin.(One single colony of BL21(DE3)/pET-gel that was grown on a LB plate in the presence of 80 μg/ml kanamycin was picked and inoculated into 20 ml LB medium at 37 °C and 225 rpm overnight. This culture was used to inoculate 1 l of the same medium. The culture was incubated at 37 °C at 220 rpm until an optical density at 600 nm of 0.7 was reached. IPTG was added to give a final concentration of 1 mM and the culture was incubated for 3 h at identical conditions. The cells were harvested by centrifugation at 6400g for 30 min at 4 °C. The pellets were suspended in 50 ml of 20 mM phosphate buffer containing 20 mM imidazole, 500 mM sodium chloride, and 1.5 mM PMSF, pH 7.2. The suspension was sonified 15 times for 8 s each in an ice-water bath. ) |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM phosphate buffer containing 100 mM imidazole, 500 mM sodium chloride, and 1.5 mM PMSF, pH 7.2 |
| Solubilization Buffer | 6 M GdnHCl, 20 mM Tris–HCl, 5 mM DTT, and 2 mM EDTA, pH 8.0 |
| Refolding Buffer | 20 mM Tris–HCl, 2 mM EDTA, 4 mM GSH, and 0.4 mM GSSG, pH 8.0 |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 16 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 4/0.4 mM |
| Refolding Protocol | The pellets from 1 l culture were suspended in 30 ml of 50 mM phosphate buffer, containing 1 mM PMSF, and 20 mg lysozyme (25,000 U), pH 7.2. The mixture was gently agitated at room temperature for 1.5 h. Then, DTT was added to a final concentration of 2 mM and the cells were disrupted five times for 30 s each by sonication in an ice-water bath. The insoluble parts of the mixture were precipitated by centrifugation at 20,000g for 30 min at 4 °C. The pellet was suspended in 40 ml B-PER reagent (Pierce, Rockford, IL, USA), that had been diluted tenfold using PBS. Again, the insoluble parts were collected by centrifugation under the same conditions. The pellet mainly contained inclusion bodies which were washed three more times as described above and were stored at −20 °C. Solubilization of inclusion bodies was achieved by incubation in 6 M GdnHCl, 20 mM Tris–HCl, 5 mM DTT, and 2 mM EDTA, pH 8.0, for 2 h at room temperature. The insoluble proteins were removed by centrifugation for 30 min at 20,000g. The denatured, solubilized fusion protein was purified by size-exclusion chromatography using a Sephacryl S-200 column (2.2 × 100 cm). The column was equilibrated with 5 M GdnHCl, 20 mM Tris–HCl, pH 8.0 (column buffer). The denatured fusion protein (11 mg/ml) was loaded onto the column. The column was eluted with column buffer at a flow rate of 0.5 ml/min. Fractions containing the fusion protein were pooled and sodium chloride and imidazole were added to a final concentration of 0.5 M and 20 mM, respectively. The fusion protein was loaded on a HiTrap Chelating HP affinity column, preloaded with nickel ions (1 M NiSO4) and equilibrated with binding buffer (5 M GdnHCl, 0.5 M NaCl, 20 mM imidazole, and 20 mM Tris–HCl, pH 8.0). The pure fusion protein was eluted with 5 M GdnHCl, 0.5 M NaCl, 200 mM imidazole, and 20 mM Tris–HCl, pH 8.0. Further washing steps were not required. Based on the methods reviewed by Rudolph and Lilie [25], a refolding protocol for the fusion protein was developed. The denatured fusion protein was diluted with refolding buffer (20 mM Tris–HCl, 2 mM EDTA, 4 mM GSH, and 0.4 mM GSSG, pH 8.0) to a final concentration of 100 μg/ml under rapid vortexing at room temperature. Refolding occurred within about 16 h under gentle stirring. Usually, about 250 μg of refolded protein was obtained from 1 mg of denatured protein. Precipitates were recycled under the same conditions after complete denaturation by 5 M GdnHCl in binding buffer as described above, but containing an additional 30 mM DTT. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |