Refolding Record:
| Protein | |
|---|---|
| Protein Name | Onconase |
| Abbreviated Name | ONC |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rana pipiens (Northern leopard frog) |
| UniProt Accession | P22069 |
| 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 | 104 |
| Molecular Weight | 11844.6 |
| Pi | 8.95 |
| Molecular Weight | 11844.6 |
| Disulphides | 4 |
| Full Sequence |
QDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Schulenburg C, Martinez-Senac MM, Löw C, Golbik R, Ulbrich-Hofmann R, Arnold U. (2007) Febs Journal, 274, 5826-5833 |
| 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 | 4 h |
| Expression Vector | pET26b |
| Expression Protocol | The experimental procedures were performed as described previously [10]. Briefly, a culture of Escherichia coli strain BL21(DE3) (Stratagene, Heidelberg, Germany) that had been transformed with a pET-26b(+) plasmid directing the expression of the ONC protein was grown in Terrific broth containing 50 µg·mL−1 kanamycin at 37 °C. Four hours after induction with 1 mm isopropyl thio-β-d-galactoside, cells were harvested. Cell lysis was performed by treatment with lysozyme and homogenization with a Gaulin homogenizer (APV Gaulin GmbH, Lübeck, Germany). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 200 mm sodium phosphate buffer (pH 7.0) |
| Refolding Buffer | 5.5 to 0.5 m guanidine hydrochloride |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 5.5 |
| Refolding Temperature | 20.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a,n/a |
| Refolding Protocol | The inclusion bodies were isolated and resolubilized. After renaturation, ONC was purified on a SOURCE S column (Amersham Biosciences, Uppsala, Sweden; 50 mm Tris/HCl, pH 7.5, with a linear gradient of 0–500 mm NaCl). Cyclization of the N-terminal glutamine residue was achieved by dialysis of the purified protein against 200 mm sodium phosphate buffer (pH 7.0) for 3 days at room temperature. The protein concentration was determined using an absorption coefficient of ε = 0.87 mL·mg−1·cm−1 at 280 nm [12]. Determination of the molecular mass to verify cyclization of the N-terminal pyroglutamate was performed by ESI (Esquire) or MALDI MS (Reflex; both from Bruker Daltonics, Bremen, Germany) after desalting of the protein samples using ZipTip pipette tips (Millipore, Schwalbach, Germany).Refolding experiments followed by stopped-flow fluorescence spectroscopyRefolding was followed by stopped-flow fluorescence spectroscopy using an SX20 stopped-flow spectrometer (Applied Photophysics Ltd, Leatherhead, UK). Emission was recorded as integral fluorescence using a 320 nm cut-off filter with excitation at 280 nm at 20 °C. Refolding was initiated by 11-fold dilution of denatured protein samples (in 6.0 m guanidine hydrochloride) into lower concentrations of denaturant. The final protein concentration was typically 5 µm, and final buffer conditions were 100 mm sodium acetate buffer (pH 5.5), containing concentrations of guanidine hydrochloride as indicated. Kinetic traces were fitted to either single, double or triple exponential equations. Amplitudes and rate constants were usually the average from three to five measurements. To assess artefacts due to the rapid mixing, control reactions were carried out using a solution of N-acetyl-l-tryptophanamide in the same molar concentration as the Tryp residues in the protein solution (5–10 µm) under the same experimental conditions as described above for the folding experiments. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |