Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prolactin |
| Abbreviated Name | Prolactin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01236 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | Mutant (1-13 residues deleted) |
| Chain Length | 183 |
| Molecular Weight | 21317.4 |
| Pi | 5.98 |
| Molecular Weight | 21317.4 |
| Disulphides | 2 |
| Full Sequence |
MTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMNQKDFLSLIVSILRSWNEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMELIVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRRDSHKIDNYLKLLKCRIIH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bernichtein, S., Jomain, J., Kelly, P. A., Goffin, V. (2003) Mol Cell Endocrinol, 208, 11-21 |
| 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 | 4h |
| Expression Vector | pT7L |
| Expression Protocol | Cells grown in IL culture, induced, and harvested via centrifugation. Cells were then lyzed and insoluble inclusion bodies isolated by further centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7-0.9 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea |
| Refolding Buffer | 50mM NH4HCO3, pH 8.0 |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 72h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were solubilized in solubilization buffer, and incubated for 5min at 55degC then for 4h at room temperature. Refolding was preformed by continuous dialysis, against refolding buffer. Protein was then concentrated by tangential-flow ultrafiltration. Centrifugation was then preformed after which supernatant was purified by gel filtration chromatography using S-200 column equilibrated with 50mM NH4HCO3 and 150mM NaCL, pH 8.0. Fractions containing protein were then pooled and dialyzed to remove NaCl, before being loaded onto a HiTrap Q anion exchange column equilibrated with 20mM TrisHCl, pH 8.0 |
| Refolding Assay | Far-UV Circular Dichroism,Bioactivity,Ligand Binding,SDS-PAGE,Isoelectic focusing |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |