Refolding Record:
| Protein | |
|---|---|
| Protein Name | Leptin |
| Abbreviated Name | Leptin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P41159 |
| 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 | 147 |
| Molecular Weight | 16097.5 |
| Pi | 5.72 |
| Molecular Weight | 16097.5 |
| Disulphides | 1 |
| Full Sequence |
AVPIQKVQDD TKTLIKTIVT RINDISHTQS VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL QGSLQDMLWQ LDLSPGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Raver N, Vardy E, Livnah O, Devos R, Gertler A (2002) General and Comparative Endocrinology, 126, 52-58 |
| 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 | 500ml of cells in Terrific Broth medium in 3L flasks was incubated at 37degC until A600 reached 0.9, at which time 50mg nalidixic acid was added to each flask. Cells were incubated a further 4h, then harvested by centrifugation (5min, 10000g) 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 | 1% Triton X-100 |
| Solubilization Buffer | 10mM Tris, 4.5M urea |
| Refolding Buffer | 10mM Tris |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 11.3 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 6h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Cells were thawed and resuspended by homogenization 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 then sonicated twice in distilled water, and then twice with 1% Triton X-100. The inclusion bodies were then dissolved in 150ml solubilization buffer. The pH was increased to 11.3 with NaOH and cysteine was added to 1mM. The solution was gently stirred at 4degC for 6h, then diluted with 3 volumes of 0.67M L-arginine and dialyzed for 48h against 5x10L of 10mM TrisHCl. The protein was then purified using a Q-sepharose column in 10mM TrisHCl pH 8.0, followed by a SP-sepharose column. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | 95% |
| Notes | n/a |