Refolding Record:
| Protein | |
|---|---|
| Protein Name | Blue copper-binding protein |
| Abbreviated Name | BCB |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Arabidopsis thaliana |
| UniProt Accession | Q07488 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | cuprodoxin domain (Glu25 to Gly130) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 89 |
| Molecular Weight | 9667.0 |
| Pi | 5.84 |
| Molecular Weight | 9667.0 |
| Disulphides | Unknown |
| Full Sequence |
YTTWATGKTFRVGDELEFDFAAGRHDVAVVSEAAFENCEKEKPISHMTVPPVKIMLNTTGPQYFICTVGDHCRFGQKLSITVVAAGATG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Harrison MD, Dennison C. (2004) Proteins: Structure, Function, and Genetics, 55, 426-435 |
| Project Aim | Expression and charactrization |
| 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 | pET11a |
| Expression Protocol | E. coli BL21(DE3) cells were transformed with pMHCD1.2 and protein expression was induced in cells grown at 37°C to an A595nm of 0.7 by the addition of 1 mM IPTG. CuSO4 (0.5 mM) was added to the culture and the cells were allowed to grow for 4 h, before harvesting by centrifugation. Typically, cell pellet from 2 L of culture was disrupted by sonication and the inclusion bodies (which contain the majority of rBCB) were collected by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.7 = 595 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 1 M urea |
| Solubilization Buffer | 25 mM Tris pH 7.5 containing 6 M urea, 10 mM DTT, and 10 mM EDTA |
| Refolding Buffer | 25 mM Tris pH 7.5, 0.5 mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The inclusion bodies were washed with 1 M urea and dissolved in 100 mL of 25 mM Tris pH 7.5 containing 6 M urea, 10 mM DTT, and 10 mM EDTA for 40 min at room temperature. rBCB was refolded at 4°C by 10-fold slow dilution (20 mL h-1) with 25 mM Tris pH 7.5, 0.5 mM EDTA, as described for recombinant CST.[30] The protein solution was diluted to 10 L with 10 mM Tris pH 7.5 supplemented with CuSO4 (0.1 mM). Refolded rBCB was captured by batch binding onto DEAE-Sepharose (Amersham Pharmacia Biotech) and eluted with 25 mM Tris pH 7.5 containing 2 M NaCl. CuSO4 (0.5 mM) was added to rBCB, the excess salt and copper were removed by dialysis, and the protein was applied to a DEAE-Sepharose column equilibrated in 25 mM Tris pH 7.5. Refolded rBCB was eluted with a 0 to 0.75 M NaCl gradient in 25 mM Tris pH 7.5 and further purified by gel filtration chromatography (Superdex 75, Amersham Pharmacia Biotech) in 25 mM Tris pH 7.5 plus 200 mM NaCl. The A280/A605 ratio, which corresponds to a single band on an SDS-PAGE gel, is 3.64 for fully oxidized rBCB. |
| Refolding Assay | Mass spectrometry,NMR analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |