Refolding Record:
| Protein | |
|---|---|
| Protein Name | CD59 glycoprotein |
| Abbreviated Name | CD59 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | O45037 |
| 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 | 80 |
| Molecular Weight | 8936.2 |
| Pi | 8.09 |
| Molecular Weight | 8936.2 |
| Disulphides | 5 |
| Full Sequence |
LRCYNCLDPVSSCKTNSTCSPNLDACLVAVSGKQVYQQCWRFSDCNAKFILSRLEIANVQYRCCQADLCNKSFEDKPNN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fraser DA, Harris CL, Williams AS, Mizuno M, Gallagher S, Smith RA, Morgan BP. (2003) Biologycal Chemistry, 278, 48921-48927 |
| Project Aim | Protein refolding |
| Fusion | c-terminal cysteine |
| 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 | pET26b |
| Expression Protocol | The sCD59-Cys cDNA in pET26b was transformed into electrocompetent E. coli BL21 (DE3) bacteria (Novagen, Nottingham, UK) by electroporation (GenePulserTM; Bio-Rad). Cells were then plated onto LB agar containing kanamycin (50 µg/ml) to select for clones containing the plasmid. Positive colonies were picked and expanded, and the presence of insert was confirmed by PCR screening. A single positive colony was picked into 25 ml of LB broth containing kanamycin (50 µg/ml) and grown overnight at 37 °C in a shaking incubator. A 20-ml aliquot of this starter culture was inoculated into 2 liters of NZCYM medium containing kanamycin (50 µg/ml) in a Bioflo 3000 Bioreactor (New Brunswick Scientific, Edison, NJ) with a 2-liter bioreactor culture vessel. The fermenter was prepared and run as described previously (18). Cultures were grown for 4 h until the bacteria were in their log phase of growth (A600 = 5–8). Protein expression was then induced by adding sterile filtered isopropyl -D-thiogalactopyranoside to a final concentration of 1 mM. The fermentation culture was harvested at 3 h post-induction and centrifuged at 10,000 x g for 10 min. Cell pellets were stored at –40 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 5-8 = 600 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris, 1 mM EDTA, 2% sodium deoxycholate (w/v), pH 8.0 |
| Solubilization Buffer | 8 M urea, 20 mM Tris, 1 mM EDTA, 50 mM 2-mercaptoethanol, pH 8.5 |
| Refolding Buffer | 50 mM Tris, 1 M NaCl, 1 mM reduced glutathione, 3 mM oxidized glutathione, pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 1 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/3 mM |
| Refolding Protocol | The cell pellet obtained above was thawed, resuspended in lysis buffer (50 mM Tris, 1 mM EDTA, 50 mM NaCl, pH 8.0; 5 ml/g of pellet), and disrupted by two passes at pressure >12,000 p.s.i. through an Emulsiflex C5 High Pressure Homogenizer (Glen Creston, Stanmore, UK) at 4 °C. The homogenate was immediately centrifuged (10,000 x g, 10 min, 4 °C), and the pellet containing sCD59-Cys in inclusion bodies was washed three times by centrifugation in 50 mM Tris, 1 mM EDTA, 2% sodium deoxycholate (w/v), pH 8.0, before being resuspended to 5 mg/ml in solubilization buffer (8 M urea, 20 mM Tris, 1 mM EDTA, 50 mM 2-mercaptoethanol, pH 8.5). Conditions for refolding of sCD59-Cys were chosen empirically by testing a large panel of solutions differing in their pH, presence of reducing and denaturing agents, presence of detergents, etc. as previously described (18, 24). Successful refolding was initially assessed by demonstrating the acquisition of epitopes recognized by the conformation-dependent mAb 6D1 in dot blots. In the chosen protocol, sCD59-Cys was refolded from solubilized inclusion bodies by rapid dilution (1:40) into 50 mM Tris, 1 M NaCl, 1 mM reduced glutathione, 3 mM oxidized glutathione, pH 8.0. The refold mixture was incubated for 1 h at 4 °C and then buffer-exchanged into PBS and concentrated 10-fold by ultra-filtration (Amicon stirred cell; 5-kDa cut-off membrane; Amicon Inc., Beverley, MA). Aggregated and/or misfolded protein was removed by ammonium sulfate precipitation. Protein in PBS was diluted 1:2.5 into 3.8 M ammonium sulfate, 0.1 M sodium phosphate buffer, pH 6.5, incubated for 10 min at room temperature, and centrifuged. The supernatant was retained and dialyzed into PBS prior to measurement of protein concentration. The molecular mass of the final product was measured using matrix-assisted laser desorption and ionization time-of-flight spectroscopy (MALDI-TOF; Bruker, Coventry, UK). The identity of the final product was confirmed by N-terminal sequencing (Applied Biosystems Procise sequencer). |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | ~5 mg |
| Purity | n/a |
| Notes | n/a |