Refolding Record:
| Protein | |
|---|---|
| Protein Name | Serine-repeat antigen protein |
| Abbreviated Name | SERA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Leishmania donovani |
| UniProt Accession | Q9NB19 |
| 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 | 102 |
| Molecular Weight | 11906.6 |
| Pi | 9.67 |
| Molecular Weight | 11906.6 |
| Disulphides | Unknown |
| Full Sequence |
MSNRFFQKFYLRCGNCTAIQRSAQGYKPIANPILFKSDEHCRNYHDEQRRAAGYAGMMVTTRCDKCNRVHSNWKVLDAQEFLDVKLSLTPAERTKRLWASSK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kothari H, Kumar P, Singh N. (2006) Protein Expression and Purification, 45, 15-21 |
| Project Aim | Drug Studies |
| Fusion | C-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 | 16 h |
| Expression Vector | pET28a |
| Expression Protocol | A single transformed BL21 (DE3) colony harboring the recombinant construct was used to prepare the preinoculum in LB broth containing 30 μg/ml kanamycin in a flask overnight at 37 °C. The preinoculum (1% v/v) was used to grow 1 L culture of cells in LB media with 30 μg/ml kanamycin at 37 °C to OD600 of 0.6–0.8, followed by isopropyl-β-d-thiogalactopyranoside induction (final concentration 0.1 mM) for 16 h at 20 °C. The cells were harvested by centrifugation at 6500 rpm for 10 min. The cell pellet was resuspended in 50 ml of buffer (20 mM sodium monobasic, pH 8.0, 200 mM NaCl, 10 mM imidazole, 0.3% N-lauryl sarcosine, 1 mM EDTA, and 1 mM PMSF). The cell suspension was sonicated (10× 30 s, Sonicator Heat system with 50% duty cycle) on ice. The resulting cell lysate was centrifuged at 14,000 rpm for 30 min. The clear supernatant was collected and used for protein purification. The pellet after centrifugation was used for inclusion body isolation. The pellet of inclusion body obtained above was washed one time each in buffers B, C, and D by centrifuging at 8000 rpm for 20 min at 4 °C. Buffer B: 20 mM sodium monobasic (pH 8.0), 1 mM EDTA, 2 M urea, 200 mM NaCl. Buffer C: 20 mM sodium monobasic (pH 8.0), 1 mM EDTA, 0.5% Triton X-100, 200 mM NaCl. Buffer D: 20 mM sodium monobasic (pH 8.0), 1 mM EDTA, 200 mM NaCl. The purified inclusion bodies were solubilised in 40 ml of 20 mM sodium monobasic (pH 8.0), 200 mM NaCl, 8 M urea, 0.5% Triton X-100, 1 mM β-mercaptoethanol, 10 mM imidazole and 10% glycerol and kept at 24 °C for 2 h. It was then centrifuged at 11,000 rpm at 4 °C for 30 min to remove the debris. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | Three washing buffer see refolding notes |
| Solubilization Buffer | 40 ml of 20 mM sodium monobasic (pH 8.0), 200 mM NaCl, 8 M urea, 0.5% Triton X-100, 1 mM β-mercaptoethanol, 10 mM imidazole and 10% glycerol |
| Refolding Buffer | 20 mM sodium monobasic (pH 8.0), 200 mM NaCl, 10 mM imidazole, and 8 M urea |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The recombinant protein was purified based on its His6-tag by affinity chromatography using a Ni2+-IDA Hi-Trap chelating Sepharose column (Amersham Biosciences). For purification of the recombinant protein from the insoluble fraction, 5 ml of the solubilised inclusion bodies, after passing through the 0.45 μm filter, was loaded onto pre-equilibrated column with buffer E (20 mM sodium monobasic (pH 8.0), 200 mM NaCl, 10 mM imidazole, and 8 M urea). The column was washed with three column volumes of buffer E containing 10 and 30 mM imidazole. Protein was eluted with 200 mM imidazole in buffer E. Fractions were collected and the presence of recombinant protein in the eluted fractions was confirmed by 12% SDS–PAGE analysis and Western blotting using anti-His monoclonal Ab. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Buffer B: 20 mM sodium monobasic (pH 8.0), 1 mM EDTA, 2 M urea, 200 mM NaCl. Buffer C: 20 mM sodium monobasic (pH 8.0), 1 mM EDTA, 0.5% Triton X-100, 200 mM NaCl. Buffer D: 20 mM sodium monobasic (pH 8.0), 1 mM EDTA, 200 mM NaCl. |