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 | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | N-terminal truncated variant bPL-DES18 |
| Chain Length | 182 |
| Molecular Weight | 20930.7 |
| Pi | 6.42 |
| Molecular Weight | 20930.7 |
| Disulphides | 2 |
| Full Sequence |
AQSLFE RATLVASNNY RLAREMFNEF NKQFGEGKNF TSKVINSCHT EFMTTPNNKE AAANTEDEAL LRLVISLLHS WDEPLHQAVT ELLHRNGASP DILARAKEIE DKTKVLLEGV EMIQKRVHPG EKKNEPYPVW SEKSSLTADD EDVRQTAFYR MFHCLHRDSS KISTYINLLK CRFTPC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gertler A, Hauser SD, Sakal E, Vashdi D, Staten N, Freeman JJ, Krivi GG (1992) J Biol Chem, 267, 12655-12659 |
| 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 | W3110 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | not stated |
| Expression Protocol | 1.5L bench-top fermentations were performed, protein production was induced with nalidixic acid for |
| Method of Induction | Nalidixic Acid |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 4.5M Urea, 10mM TrisHCl pH 8.0 |
| Refolding Buffer | 40mM TrisHCl, 0.1mM cysteine, pH 11.3 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 11.3 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 48h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 0.1mM |
| Refolding Protocol | Pelleted cells (6-10g) were thawed and resuspended in 10mM EDTA pH 8.0 in the presence of 0.5mg/ml lysozyme. Following 30min incubation, the cells were sonicated and pelleted (30min, 25000g). The pellet was sonicated twice in distilled water then pelleted and dissolved in solubilization buffer. After removal of insoluble material, the concentration wof TrisHCl was raised to 40mM, the pH increased to 11.3 with NaOH and cystein was added to 0.1mM. After stirring at 48h at 4degC, the sution was loaded onto a monoQ column equilibrated with 10mM TrisHCl at pH 9.0. The protein was eluted using a discontinuous NaCl gradient, selected fractions were dialyzed against 0.05% NaHCO3 and freeze-gried. |
| Refolding Assay | Far-UV Circular Dichroism,Bioactivity,SDS-PAGE,Gel filtration chromatography,Receptor binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |