Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ribonuclease mitogillin also known as Restrictocin |
| Abbreviated Name | pRest |
| SCOP Family | Ribotoxin |
| Structure Notes | |
| Organism | Aspergillus restrictus |
| UniProt Accession | P67876 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 150 |
| Molecular Weight | 16859.8 |
| Pi | 9.12 |
| Molecular Weight | 16859.8 |
| Disulphides | Unknown |
| Full Sequence |
ATWTCINQQLNPKTNKWEDKRLLYSQAKAESNSHHAPLSDGKTGSSYPHWFTNGYDGNGKLIKGRTPIKFGKADCDRPPKHSQNGMGKDDHYLLEFPTFPDGHDYKFDSKKPKEDPGPARVIYTYPNKVFCGIVAHQRGNQGDLRLCSH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Goyal A, Seth D, Batra JK. (2002) Biochem Biophys Res Commun., 295, 812-817 |
| Project Aim | Undefined |
| Fusion | T7 |
| 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 | 2 h |
| Expression Vector | n/a |
| Expression Protocol | Expression and purification of proteins. E. coli BL21 (λDE3) cells were transformed with the desired construct and grown in Super broth containing 100 μg/ml ampicillin, at 37 °C. The cultures were induced at A600 of 2.0 with 1 mM IPTG and harvested 2 h later. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | guanidine hydrochloride |
| Refolding Buffer | arginine and oxidised glutathione |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The recombinant proteins were purified from the inclusion bodies as described by Buchner et al. [21]. The purified inclusion bodies were solubilized in guanidine hydrochloride, reduced by dithioerythritol and recombinant proteins renatured in a buffer containing arginine and oxidised glutathione. Renatured material, after dialysis in 20 mM MES buffer, pH 5.0 was loaded on a S-Sepharose column, and the bound proteins were eluted with a 0–1 M NaCl gradient using an FPLC system (Pharmacia). The proteins were further purified to homogeneity by gel filtration chromatography on TSK 3000 column. |
| Refolding Assay | Cytotoxicity Assays |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |