Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ribonuclease A |
| Abbreviated Name | RNase A |
| SCOP Family | Ribonuclease A-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07998 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 128 |
| Molecular Weight | 14577.3 |
| Pi | 8.83465 |
| Molecular Weight | 14577.3 |
| Disulphides | 4 |
| Full Sequence |
ME SRAKKFQRQH MDSDSSPSSS STYCNQMMRR RNMTQGRCKP VNTFVHEPLV DVQNVCFQEK VTCKNGQGNC YKSNSSMHIT DCRLTNGSRY PNCAYRTSPK ERHIIVACEG SPYVPVHFDA
SVEDST
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Canals A, Ribo M, Benito A, Bosch M, Mombelli E, Vilanova M (1999) Protein Expression and Purification, 17, 169-181 |
| Project Aim | Structure-Function |
| Fusion | None |
| 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 | 3-4h |
| Expression Vector | pSR1 |
| Expression Protocol | LB medium (with 0.4mg/ml ampicillin) was inoculated with 1:100 overnight culture and grown at 37degC with agitation. Protein expression was induced with 1mM IPTG when A550 reached 1-2. Cells were grown for a further 3-4h and harvested by centrifugation (20min x 10000g, 4degC). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 550 = 1-2 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl, 100mM Tris-acetate, 2mM EDTA, pH 8.5 |
| Refolding Buffer | 100mM Tris-acetate, 0.5M L-arginine, 8mM GSSG, 2mM EDTA pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 10.0 |
| Protein Concentration | 0.05-0.1mg/ml |
| Refolding Time | >24hr |
| Redox Agent | GSSG |
| Redox Agent Concentration | 8mM,8mM |
| Refolding Protocol | Cell pellets from 1L induced cultures were resuspended in 30ml for 50mM TrisHCl, 10mM EDTA pH 8.0, sonicated and centrifuged (10min x 12000g, 4degC). Pellets containing about 75mg of protein were resuspended in solubilization buffer, samples were then reduced by the addition of 0.1M GSH, the pH was adjusted to 8.5 with solid Tris, and the sample was then incubated at room temperature under nitrogen atmosphere for 2h. The sample was then centrifuged (30min x 12000g) and the supernatant was diluted approx. 100-fold to a final concentration of 0.05-0.1mg/ml into refolding buffer and incubated at 10degC for at least 24h. The pH was adjusted to 5.0 with acetic acid, then the sample was concentrated by ultrafiltration and dialyzed exhaustively against 50mM sodium acetate pH 5.0. Insoluble material was removed by centrifugation (12000g x 10min, 4degC), and the protein was then purified using a Mono-S column with a linear gradient from 0 to 600mM NaCl. Fractions containing pure protein were dialyzed against ultrapure water and lyophilized. |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | 5mg/L culture |
| Purity | |
| Notes | |