Refolding Record:
| Protein | |
|---|---|
| Protein Name | Leptin |
| Abbreviated Name | Leptin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Pig (Sus scrofa) |
| UniProt Accession | Q29406 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 147 |
| Molecular Weight | 16145.7 |
| Pi | 6.32 |
| Molecular Weight | 16145.7 |
| Disulphides | 1 |
| Full Sequence |
AVPIWRVQDD TKTLIKTIVT RISDISHMQS
VSSKQRVTGL DFIPGLHPVL SLSKMDQTLA IYQQILTSLP SRNVIQISND LENLRDLLHL LASSKSCPLP QARALETLES LGGVLEASLY STEVVALSRL QGALQDMLRQ LDLSPGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Raver N, Gussakovsky EE, Keisler DH, Krishna R, Mistry J, Gertler A (2000) Protein Expression and Purification, 19, 30-40 |
| 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 | MON105 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pTRC |
| Expression Protocol | Cells were grown in TB medium at 37degC. When A600 reached 1.0, 25mg of freshly prepared nalidix acid dissolve in 2.5ml 0.1N NaOh was added. Bacteria were incubated for an additional 4h then harvested by centrifugation (10000g) and frozen at -20degC. |
| Method of Induction | Nalidixic Acid |
| Cell Density at Induction | OD 600 = 1.0 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 1% Triton X-100 |
| Solubilization Buffer | 4.5M urea, 10mM Tris, 1mM cystein pH 11.3 |
| Refolding Buffer | 10mM TrisHCl pH 8.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 48h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Cells were thawed and resuspended by homogneization in 10mM EDTA pH 8.0 with 0.5mg/ml lysozyme. Following 30min incubation, the cells were sonicated and centrifuged (30min, 25000g). The pellet was sonicated twice in distilled water then centrifuged, then washed 4 more times, twice with wash buffer and twice more with water. The inclusion body pellet was then solubilized in 500ml solubilization buffer and the solution was gently stirred at 5degC for 6h, before being diluted with three volumes of 0.67M L-arginine and then dialyzed for 48h against 5x 10L of refolding buffer. |
| Refolding Assay | Far-UV Circular Dichroism,Bioactivity,SDS-PAGE,Gel filtration chromatography,Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.67M |
| Refolding Yield | 36mg/L culture |
| Purity | n/a |
| Notes | n/a |