Refolding Record:
| Protein | |
|---|---|
| Protein Name | Angiogenin-3 |
| Abbreviated Name | mAng-3 |
| SCOP Family | Ribonuclease A-like |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | P97802 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 123 |
| Molecular Weight | 14187.1 |
| Pi | 9.18371 |
| Molecular Weight | 14187.1 |
| Disulphides | 3 |
| Full Sequence |
QDNYRYIKFLTQHYDAKPTGRDYRYCESMMKKRKLT
SPCKEVNTFIHDTKNNIKAICGENGRPYGVNFRISNSRFQVTTCTHKGGSPRPPCQYNAF
KDFRYIVIACEDGWPVHFDESFISP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Holloway DE, Hares MC, Shapiro R, Subramanian V, Acharya KR (2001) Protein Expression and Purification, 22, 307-317 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)CodonPlus-RIL |
| Expression Temp | 37.0 |
| Expression Time | 2 hours |
| Expression Vector | pET-22b(+) |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | None |
| Solubilization Buffer | 7M Gdn-HCl, 0.15M GSH, 0.1M Tris-HCl, 0.002M EDTA, pH 8 |
| Refolding Buffer | 0.5M L-arginine, 0.003M GSH, 0.0006M GSSG, pH 8 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 20.0 |
| Protein Concentration | 5-10 ug/ml |
| Refolding Time | 24 hours |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Protein solubilization and purification were conducted at room temperature. The insoluble cell extract obtained from 0.5 litre bacterial culture was dispersed in ~10 ml 7 M guanidine hydrochloride, 0.15 M reduced glutathione, 0.1 M Tris-HCl, 2 mM EDTA, pH 8.0, and stirred under nitrogen for 2 h. The sample was then added dropwise with gentle stirring to 500 ml 0.5 M L-arginine HCl, pH 8.0, containing 0.6 mM oxidized glutathione, and left to stand for 24 h. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 12 mg/litre of culture |
| Purity | >95% after column chromatography |
| Notes | Construct encodes full protein. Protein construct sequence: MQDNYRYIKFLTQHYDAKPTGRDYRYCESMMKKRKLTSPCKEVNTFIHDTKNNIKAICGENGRPYGVNFRISNSRFQVTTCTHKGGSPRPPCQYNAFKDFRYIVIACEDGWPVHFDESFISP Difference from genomic sequence: additional Met at N-terminus. |