Refolding Record:
| Protein | |
|---|---|
| Protein Name | OriC-binding nucleoid-associated protein |
| Abbreviated Name | Cnu |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P64467 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 71 |
| Molecular Weight | 8416.6 |
| Pi | 6.24 |
| Molecular Weight | 8416.6 |
| Disulphides | Unknown |
| Full Sequence |
MTVQDYLLKFRKISSLESLEKLYDHLNYTLTDDQELINMYRAADHRRAELVSGGRLFDLGQVPKSVWHYVQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kim MS, Bae SH, Yun SH, Lee HJ, Ji SC, Lee JH, Srivastava P, Lee SH, Chae H, Lee Y, Choi BS, Chattoraj DK, Lim HM. (2005) Journal of Bacteriology, 187, 6998-7008 |
| Project Aim | Undefined |
| 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 | 6 h |
| Expression Vector | pET15b |
| Expression Protocol | The cnu gene was PCR amplified from pHL355-36 and cloned between the NdeI and BamHI sites of pET-15b (Novagen), generating pETCnu. For the expression of 6xHis-Cnu (Cnu protein tagged with six histidines at the N terminus), BL21 (DE3)/pETCnu cells were grown in M9 minimal medium containing Amp (50 mg/ml) at 37°C for 16 h. A one-tenth dilution of this culture was made in fresh M9 medium (1 liter), and cells were grown to an A600 of 0.7 at 37°C. IPTG (final concentration, 0.4 mM) was added, and the culture was grown for an additional 6 h. Cells were pelleted and resuspended in 35 ml of phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4). Phenylmethylsulfonyl fluoride (final concentration, 0.2 mM) was added. Cells were disrupted by sonication (15 cycles of 10 s on and 60 s off). The cell lysate was cleared by centrifugation (12,000 x g) for 45 min at 4°C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.7 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM sodium phosphate, pH 7.3, 200 mM NaCl, 1% Triton X-100 |
| Solubilization Buffer | 40 mM sodium acetate, pH 7.3, 400 mM NaCl containing 6 M guanidium chloride |
| Refolding Buffer | 50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 5 mM imidazole, 1 M urea, 10% D-glucose, 2 mM MgCl2 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| 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 cell lysate was cleared by centrifugation (12,000 x g) for 45 min at 4°C. The pellet was washed twice with a washing buffer (20 mM sodium phosphate, pH 7.3, 200 mM NaCl, 1% Triton X-100) and dissolved in a buffer (40 mM sodium acetate, pH 7.3, 400 mM NaCl) containing 6 M guanidium chloride. Denatured proteins were refolded by rapid dilution using a refolding buffer (50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 5 mM imidazole, 1 M urea, 10% D-glucose, 2 mM MgCl2). We kept the final concentration of proteins below an A280 of 0.2/ml. The resulting protein solution was centrifuged (8,000 x g) for 30 min, and the supernatant was loaded onto a 5 ml Ni-NTA Superflow column (QIAGEN) and preequilibrated with the binding buffer (50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 5 mM imidazole). The column was washed with 50 ml of the binding buffer, followed by 30 ml of the washing buffer (50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 50 mM imidazole), and the target protein (6xHis-Cnu) was eluted from the column with 50 ml of the elution buffer (50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 1 M imidazole). Fractions containing the target protein were pooled and concentrated to 10 ml using a stirred concentration cell (Amicon). Nuclear magnetic resonance spectroscopic study of the refolded 6xHis-Cnu and the native Cnu indicated that there are no structural differences between these two proteins (data not shown). |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |