Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prolactin |
| Abbreviated Name | Prolactin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Ovis aries (sheep/ovine) |
| UniProt Accession | P01240 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 229 |
| Molecular Weight | 25777.6 |
| Pi | 6.23 |
| Molecular Weight | 25777.6 |
| Disulphides | 3 |
| Full Sequence |
MDSKGSAQKG SRLLLLLVVS NLLLCQGVVS TPVCPNGPGN CQVSLRDLFD RAVMVSHYIH NLSSEMFNEF DKRYAQGKGF ITMALNSCHT SSLPTPEDKE QAQQTHHEVL MSLILGLLRS WNDPLYHLVT EVRGMKGVPD AILSRAIEIE EENKRLLEGM EMIFGQVIPG AKETEPYPVW SGLPSLQTKD EDARHSAFYN LLHCLRRDSS KIDTYLKLLN CRIIYNNNC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Leibovich H, Raver N, Herman A, Gregoraszczuk EL, Gootwin E, Gertler A (2001) Protein Expression and Purification, 22, 489-496 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | MON105 |
| Expression Temp | 37.0 |
| Expression Time | 12-14h |
| Expression Vector | pMON3401 |
| Expression Protocol | Cells were grown in 5x500ml Terrific Broth medium. When OD600 reached 0.9, protein expression was induced by the addition of 50microg/ml nalidixic acid. Cells were grown for a further 12-14h then harvested by centrifugation (10min x 10000g). |
| Method of Induction | Nalidixic Acid |
| Cell Density at Induction | OD 600 = 0.9 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 1% Triton-X |
| Solubilization Buffer | 4.5M urea, 10mM Tris, 1mM cysteine pH 11.3 |
| Refolding Buffer | 10mM TrisHCl, pH 9 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 48 |
| Redox Agent | Cysteine |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The bacterial pellet was resuspended in 200ml of 10mM EDTA pH 8, containing 100mg lysozyme and mixed gently on ice for 30min. The suspension was then sonicated on ice and the inclusion bodies were collected by centrifugation (15000g x 15min). The inclusion bodies were then twice resuspended in distilled water, sonicated and centrifuged, then washed a further two times in wash buffer followed by another two washes with water. The pellet from one batch of culture (500ml culture) was then dissolved in 500ml solubilization buffer and the solution gently stirred at 4degC for 1h. The solution was then diluted with 2 volumes of cold water and dialyzed for 48h against 5 x 10L of refolding buffer. The refolded protein was then loaded onto a Q-sepharose column preequilibrated with refolding buffer, the protein was eluted using a discontinuous NaCl gradient in 10mM TrisHCl pH 9. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 52% recovery |
| Purity | |
| Notes | |