Refolding Record:
| Protein | |
|---|---|
| Protein Name | Chitinase A1 |
| Abbreviated Name | CHA1 |
| SCOP Family | Type II chitinase |
| Structure Notes | |
| Organism | Bacillus circulans |
| UniProt Accession | P20533 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Chitin-binding domain (CHBD) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 44 |
| Molecular Weight | 4964.6 |
| Pi | 8.14 |
| Molecular Weight | 4964.6 |
| Disulphides | Unknown |
| Full Sequence |
WQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ferrandon S, Sterzenbach T, Mersha FB, Xu MQ. (2003) Biochemica et Biophysica Acta, 1621, 31-40 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | ER2566 |
| Expression Temp | 0.0 |
| Expression Time | 3 h |
| Expression Vector | pGM-CSF-ChBD |
| Expression Protocol | E. coli ER2566 cells carrying pGM-CSF-ChBD or pHer-2(KD)-ChBD were induced for 3 h in the presence of 0.3 mM IPTG after cell density had reached an A600 of 0.5–0.7. Induced cells were collected by centrifugation and resuspended in 20 mM Tris–HCl (pH 8) containing 0.5 M NaCl (Buffer A). Both fusion proteins were found in inclusion bodies that were isolated by breaking cells by sonication followed by centrifugation at 15,000×g for 30 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-0.7 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mM Tris–HCl (pH 8) containing 0.5 M NaCl (Buffer A) |
| Solubilization Buffer | 20 mM Tris–HCl (pH 8) containing 0.5 M NaCl, 7 M guanidine–HCl and 10 mM DTT |
| Refolding Buffer | Buffer A containing 8 M urea, 10 mM DTT; Buffer A containing 6 M urea, 1 mM DTT; Buffer A containing 4 M urea, 1 mM DTT; and, in the final step, Buffer A containing 2 M urea, 0.1 mM oxidized glutathione and 1 mM reduced glutathione |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG/DTT |
| Redox Agent Concentration | 1/0.1/1 mM,1/0.1/1 mM,1/0.1/1 mM |
| Refolding Protocol | The proteins were solubilized in 20 mM Tris–HCl (pH 8) containing 0.5 M NaCl, 7 M guanidine–HCl and 10 mM DTT. Insoluble components were removed by centrifugation at 15,000×g for 30 min. The fusion proteins was renatured by dialyzing the supernatant successively at 4 °C against the following buffers: Buffer A containing 8 M urea, 10 mM DTT; Buffer A containing 6 M urea, 1 mM DTT; Buffer A containing 4 M urea, 1 mM DTT; and, in the final step, Buffer A containing 2 M urea, 0.1 mM oxidized glutathione and 1 mM reduced glutathione. Renatured fusion proteins were dialyzed twice against 20 mM Tris–HCl, 0.5 M NaCl containing 0.1 mM oxidized glutathione, and 1 mM reduced glutathione. Insoluble components were removed by centrifugation at 15,000×g for 30 min and the final NaCl concentration was increased to 2 M. Binding was performed by loading the supernatant onto a 25 ml chitin resin equilibrated in 20 mM Tris–HCl (pH 8) containing 2 M NaCl. The column was washed with 30 column volumes of the same buffer and then flushed with the elution buffer containing 20 mM Tris–HCl (pH 8) and 50 mM NaCl. Proteins were analyzed on a 12% Tris–glycine gel. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |