Refolding Record:
| Protein | |
|---|---|
| Protein Name | Nuclease |
| Abbreviated Name | NucA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Anabaena sp. |
| UniProt Accession | P38446 |
| 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 | 251 |
| Molecular Weight | 27400.6 |
| Pi | 5.47 |
| Molecular Weight | 27400.6 |
| Disulphides | Unknown |
| Full Sequence |
QVPPLTELSPSISVHLLLGNPSGATPTKLTPDNYLMVKNQYALSYNNSKGTANWVAWQLNSSWLGNAERQDNFRPDKTLPAGWVRVTPSMYSGSGYDRGHIAPSADRTKTTEDNAATFLMTNMMPQTPDNNRNTWGNLEDYCRELVSQGKELYIVAGPNGSLGKPLKGKVTVPKSTWKIVVVLDSPGSGLEGITANTRVIAVNIPNDPELNNDWRAYKVSVDELESLTGYDFLSNVSPNIQTSIESKVDN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Korn C, Meiss G, Gast F, Gimadutdinow O, Urbanke C, Pingoud A. (2000) Gene, 253, 221-229 |
| Project Aim | Recombinant Protein Expression |
| Fusion | N-terminal GST |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)Gold |
| Expression Temp | 37.0 |
| Expression Time | 2 h |
| Expression Vector | pGM-C10H6 |
| Expression Protocol | For the production of the NucA/NuiA complex by tandem overexpression of the nucA and nuiA genes, E. coli BL21Gold(DE3) cells were transformed with the respective plasmids. Proteins produced with a His6 affinity tag at the N-terminus were purified by Ni2+ NTA affinity chromatography. A 500 ml culture of transformed E. coli BL21Gold(DE3) cells was grown until an OD600 of 0.5 was reached and expression induced by the addition of IPTG to a final concentration of 1 mM. After 2 h of induction, cells were harvested and resuspended in 10 ml STE buffer (10 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange/size exclusion/HIC |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 10 mM Tris–HCl, pH 8.2 |
| Solubilization Buffer | 6 M urea, |
| Refolding Buffer | 10 mM Tris–HCl, pH 8.2, containing 0.062 g reduced glutathione |
| Pre-Refolding Purification | Ion-exchange/size exclusion/HIC |
| Tag Cleaved | yes |
| Refolding pH | 8.2 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH |
| Redox Agent Concentration | 0.062 g |
| Refolding Protocol | the pellet was resuspended in buffer A (10 mM Tris–HCl, pH 8.2) and sonicated using a Branson sonifier. The suspension was centrifuged and the presence of NucA and NuiA in the supernatant as well as in the solubilized pellet checked by SDS-PAGE. Solutions containing native proteins were applied directly to 1 ml of a Ni2+ NTA resin, whereas denatured proteins from the solubilized pellets, using buffer A containing 6 M urea, were first dialysed against buffer A and then applied to the column. The column was washed with 20 ml buffer A containing 20 mM imidazole and then with buffer A containing 500 mM NaCl. Elution was performed using buffer A supplemented with 200 mM imidazole, and eluted proteins were dialysed against buffer A containing 20% glycerol and 0.01% lubrol. The variant carrying a GST-tag at the N-terminus was purified on 1 ml glutathione-sepharose columns using 50 ml 10 mM Tris–HCl, pH 8.2, 100 mM NaCl as the washing buffer and 10 ml 10 mM Tris–HCl, pH 8.2, containing 0.062 g reduced glutathione as the elution buffer. The concentrations of the enzymes were determined by UV spectroscopy using extinction coefficients calculated according to Pace et al. (1995): 51 910 M−1 cm−1 for NucA and 16 960 M−1 cm−1 for NuiA. |
| Refolding Assay | Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |