Refolding Record:
| Protein | |
|---|---|
| Protein Name | Exo-beta-1,4-glucanase |
| Abbreviated Name | Cex |
| SCOP Family | Cellulose-binding domain family II |
| Structure Notes | |
| Organism | Cellulomonas fumi |
| UniProt Accession | Q59277 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Cellulose binding domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 100 |
| Molecular Weight | 10296.2 |
| Pi | 8.06796 |
| Molecular Weight | 10296.2 |
| Disulphides | 0 |
| Full Sequence |
CQVLWGVN QWNTGFTANV TVKNTSSAPV DGWTLTFSFP SGQQVTQAWS STVTQSGSAV TVRNAPWNGS IPAGGTAQFG FNGSHTGTNA APTAFSLNGT PC
|
| Notes | Sequence does not include steptavidin tag |
| Expression | |
|---|---|
| Report | Le KD, Gilkes NR, Kilburn DG, Miller RC, Saddler JN, Warren RAJ (1994) Enzyme Microb. Technol., 16, 496-500 |
| Project Aim | Undefined |
| Fusion | N-terminal streptavidin |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4-5h |
| Expression Vector | not stated |
| Expression Protocol | 500 cultures of cells were grown, 0.4mM IPTG was added when A600 reached 1.0. After 4-5h further incubation cells were harvested. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 1.0 |
| Cell Disruption Method | Chemical |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 30mM TrisHCl pH 8.0, 1mM EDTA, 1% Triton X-100 |
| Solubilization Buffer | 10mM potassium phosphate pH 7.0, 6M GdnHCl, 1% beta-mercaptoethanol |
| Refolding Buffer | 10mM potassium phosphate pH 7.0, 0.05% beta-mercaptoethanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 16-24h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 0.05% |
| Refolding Protocol | The cells were washed by resuspension in 10mM TrisHCl pH 8.0, 100mM NaCl and 1mM EDTA. The cells were centrifuged again, then lysed by resuspension in 30mM TrisHCl pH 8.0, 2 mM EDTA and 0.1% Triton X-100. The lysate was stored at -70degC. The lysate was thawed, then MgSO4, DNasI and RNase A were added to final concentrations of 12mM , 10microg/ml and 10 microg/ml respectively and the mixture was allowed to stand for 15-20min. The suspension was then centrifuged (15min, 6000g) and the pellet was washed three times with wash buffer. The pellet was then dissolved in solubilization buffer and dialyzed against the same buffer. The solution was then slowly diluted 100-fold into refolding buffer and stirred gently at 4degC for 16-24h. The protein was then dialyzed against 40volumes of renaturation buffer with several buffer changes. The solution was then centrifuged (20min, 39000g) and the protein filtered. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |