Refolding Record:
| Protein | |
|---|---|
| Protein Name | Placental lactogen |
| Abbreviated Name | PL |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P09611 |
| 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 | 236 |
| Molecular Weight | 26908.8 |
| Pi | 6.75011 |
| Molecular Weight | 26908.8 |
| Disulphides | 2 |
| Full Sequence |
MAPASSHRGH QWICDLVRGS CLLLLLVVSN LLLCQG
VEDY APYCKNQPGN CRIPLQSLFE RATLVASNNY RLAREMFNEF NKQFGEGKNF TSKVINSCHT EFMTTPNNKE AAANTEDEAL LRLVISLLHS WDEPLHQAVT ELLHRNGASP DILARAKEIE DKTKVLLEGV EMIQKRVHPG EKKNEPYPVW SEKSSLTADD
EDVRQTAFYR MFHCLHRDSS KISTYINLLK CRFTPC
|
| Notes | According to UniProt entry, aa.1-36 is a signal sequence In this study, two analogues: G133K and G133R expressed and purified. (numbering seems to apply without the signal sequence) |
| Expression | |
|---|---|
| Report | Helman D, Staten NR, Grosclaude J, Daniel N, Nespouous C, Djiane J, Gertler A (1998) J Biol Chem, 273, 16067-16074 |
| Project Aim | Functional Studies |
| 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 | 4h |
| Expression Vector | pMON3401 |
| Expression Protocol | Cells were grown in 500ml Terrific Broth medium with shaking (200rpm) at 37degC in 2L flasks. When A600 reached 0.9, 25mg nalidixic acid was added to each flask. Cells were incubated for a further 4h, then centrifuged (5min, 10000g), decanted and frozen at -20degC. |
| 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 | Dilution/Dialysis combination |
| Wash Buffer | distilled water |
| Solubilization Buffer | 4.5M urea, 10mM Tris |
| Refolding Buffer | 10mM Tris pH 9 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 96h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 0.1mM |
| Refolding Protocol | Cells were thawed and resuspended by homogenization in 10mM EDTA pH 8.0 in the presence of 0.5mg/ml lysozyme. Following 30min incubation, cells were sonicated and centrifuged (30min, 25000g). The pellet was sonicated twice in distilled water and centrifuged. The inclusion bodies from 2.5L cell culture were then solubilized in 600ml Solubilization buffer. The pH was increased to 11.3 with NaOH, cysteine was added to 0.1mM and the clear solution was stirred at 4degC for 48h, then dialyzed for a further 48h against 5x10L of refolding buffer. The protein was then purified using a Q-sepharose column. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | See also Gertler et al (1992), J Biol Chem, v.267, p12655-12659 for more details of protocol and sequence |