Refolding Record:
| Protein | |
|---|---|
| Protein Name | Angiogenin |
| Abbreviated Name | Ang |
| SCOP Family | Ribonuclease A-like |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | Q2NKV1 |
| 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 | 148 |
| Molecular Weight | 17003.5 |
| Pi | 9.09 |
| Molecular Weight | 17003.5 |
| Disulphides | Unknown |
| Full Sequence |
MVMVLSPLFLVFILGLGLTPVAPAQDDYRYIHFLTQHYDAKPKGRNDEYCFNMMKNRRLTRPCKDRNTFIHGNKNDIKAICEDRNGQPYRGDLRISKSEFQITICKHKGGSSRPPCRYGATEDSRVIVVGCENGLPVHFDESFITPRH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jang SH, Kang DK, Chang SI, Scheraga HA, Shin HC. (2004) Biotechnol Lett, 26, 1501-1504 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DE3 |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pETbAng |
| Expression Protocol | E. coli strain Rosetta (DE3)pLysS, carrying the plasmid pETbAng, was used for expression of rbAng. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 ml 6 M guanidine hydrochloride (GdnHCl), 100 mM Tris/HCl (pH 8), 1 mM EDTA, 100 mM NaCl, 10 mM reduced DTT |
| Refolding Buffer | 0.5 mM DTTred , 0.3 M GdnHCl with 100 mM Tris/HCl (pH 8), 1 mM EDTA, 0.3 mM GSSG, 1.5 mM GSH |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1.5/3 mM |
| Refolding Protocol | Isolated inclusion bodies were dissolved in 20 ml 6 M guanidine hydrochloride (GdnHCl), 100 mM Tris/HCl (pH 8), 1 mM EDTA, 100 mM NaCl, 10 mM reduced DTT (DTTred ) and diluted for refolding to 0.2 mg protein ml−1 , 0.5 mM DTTred , 0.3 M GdnHCl with 100 mM Tris/HCl (pH 8), 1 mM EDTA, 0.3 mM GSSG, 1.5 mM GSH, and then incubated for 24 h at 4 ◦ C. Once folding was judged to be complete, the solution was concentrated by ultrafiltration and then loaded onto a cation-exchange FPLC column packed with SP-Sepharose Fast Flow (Amersham Pharmacia Biosciences) and equilibrated with 25 mM Tris/HCl (pH 8). The collected fractions from the FPLC column were dialyzed against water and lyophilized. The purified rbAng was characterized by various analytical methods, including N -terminal sequencing, mass spectrometry, circular dichroism, and bioassays, and identical to the nbAng from cow’s milk. |
| Refolding Assay | Biological assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |