Refolding Record:
| Protein | |
|---|---|
| Protein Name | Carbonic anhydrase IV |
| Abbreviated Name | CA IV |
| SCOP Family | Carbonic anhydrase |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | Q64444 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 261 |
| Molecular Weight | 29628.7 |
| Pi | 8.49 |
| Molecular Weight | 29628.7 |
| Disulphides | Unknown |
| Full Sequence |
EDSGWCYEIQTKDPRSSCLGPEKWPGACKENQQSPINIVTARTKVNPRLTPFILVGYDQKQQWPIKNNQHTVEMTLGGGACIIGGDLPARYEAVQLHLHWSNGNDNGSEHSIDGRHFAMEMHIVHKKLTSSKEDSKDKFAVLAFMIEVGDKVNKGFQPLVEALPSISKPHSTSTVRESSLQDMLPPSTKMYTYFRYNGSLTTPNCDETVIWTVYKQPIKIHKNQFLEFSKNLYYDEDQKLNMKDNVRPLQPLGKRQVFKS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hurt JD, Tu C, Laipis PJ. (1998) Protein Expression and Purification, 12, 7-16 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pET31 |
| Expression Protocol | The different forms of MCA IV were expressed in Escherichia coli strain BL21 (DE3) pLysS as previously described (46). |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2 |
| Solubilization Buffer | 5.6 M guanidine hydrochloride, 100 mM Tris HCl, pH 8.0, 1 mM EDTA, and 1 mM dithiothreitol (DTT) dissolved in degassed,sterile H2O |
| Refolding Buffer | 100 mM Tris HCl, pH 8.0, 1 mM EDTA, 40 nM DTT, 1 mM ZnCl2 , 220 mM guanidine hydrochloride |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 18 uM |
| Refolding Time | 24 h |
| Redox Agent | DTT |
| Redox Agent Concentration | 40 nM |
| Refolding Protocol | Small-scale protein refolding. An aliquot of inclusion body suspension (12 – 200 ml at 50 mg/ml protein) was placed in a 2-ml screw cap microcentrifuge tube. After centrifugation for 2 min at 10,000g, the pellet was resuspended in denaturing buffer (5.6 M guanidine hydrochloride, 100 mM Tris HCl, pH 8.0, 1 mM EDTA, and 1 mM dithiothreitol (DTT) dissolved in degassed, sterile H2O) to a final denatured protein concentration of 14 mg/ml. This solution was incubated with gentle agitation for 6 – 16 h at room temperature. Unless otherwise stated, aliquots of the denatured protein were rapidly diluted 1:25 into degassed refolding buffer [final concentrations: 100 mM Tris HCl, pH 8.0, 1 mM EDTA, 40 nM DTT, 1 mM ZnCl2 , 220 mM guanidine hydrochloride, and 0.54 mg/ml (18 mM) protein] and sealed in 2-ml screw cap microcentrifuge tubes. The protein was allowed to refold 24 h at room temperature before activity was assayed. |
| Refolding Assay | Protein activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |