Refolding Record:
| Protein | |
|---|---|
| Protein Name | Adrenomedullin |
| Abbreviated Name | AM |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P35318 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 53 |
| Molecular Weight | 6088.9 |
| Pi | 9.69587 |
| Molecular Weight | 6088.9 |
| Disulphides | Unknown |
| Full Sequence |
YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYG
|
| Notes | Thioredoxin-protein fusion, incorporating aa.95-146 (mature protein), but thioredoxin cleaved prior to refolding |
| Expression | |
|---|---|
| Report | Mitsuda Y, Takimoto A, Kamitani S, Kitamura K, Sakata T, Mitsushima K. (2002) Protein Expression and Purification, 25, 448-455 |
| Project Aim | Folding |
| Fusion | N-terminal thioredoxin |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM109/BL21 |
| Expression Temp | 37.0 |
| Expression Time | 24h |
| Expression Vector | pAME3105 |
| Expression Protocol | Cells were grown in CAH-A medium or modified TB medium (see paper for details). Cells were grown at 28degC or 37degC until OD650 reached 0.3, when 1mM IPTG was added. The cells were grown a further 24h then harvested by centrifugation |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 650 = 0.3 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM TrisHCl pH 7.0 |
| Solubilization Buffer | 6M urea, 50mM TrisHCl pH 8.0 |
| Refolding Buffer | 50mM TrisHCl pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were sonicated and inclusion bodies were collected by centrifugation, then washed twice by resuspension in Wash buffer and centrifugation. Inclusion bodies were then dissolved in solubilization buffer, and the protein was then diluted into refolding buffer. The protease BLase was added to the mixture with 1M urea, and the mixture incubated for 1h at 37degC to allow cleavage of the thioredoxin tag. The protein formed an insoluble precipitate during the cleaveage reaction, and was therfore allowed to precipitate further for 16h at 5degC. The precipiate was then washed three times with distilled water and extracted in 50mM sodium acetate (pH 4.0), recovery of the protein was improved by heating to 80degC for 30min. The protein was then amidated by oxidation (see paper). The protein was then purified by cation-exchange chromatography and RP-HPLC. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 82mg/L |
| Purity | |
| Notes | |