Refolding Record:
| Protein | |
|---|---|
| Protein Name | Leptin |
| Abbreviated Name | Leptin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Ovis aries (sheep/ovine) |
| UniProt Accession | Q28603 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | R128Q |
| Chain Length | 146 |
| Molecular Weight | 16025.6 |
| Pi | 6.31 |
| Molecular Weight | 16025.6 |
| Disulphides | 1 |
| Full Sequence |
VPIRKVQDDT KTLIKTIVTR INDISHTQSV SSKQRVTGLD FIPGLHPLLS LSKMDQTLAI YQQILASLPS RNVIQISNDL ENLRDLLHLL AASKSCPLPQ VRALESLESL GVVLEASLYS TEVVALSQLQ GSLQDMLRQL DLSPGC
|
| 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 | 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 | pTrc99A |
| Expression Protocol | Cells were grown in TB medium at 37degC. When A600 reached 2.5, 500mg of freshly prepared nalidixic acid dissolved in 50ml NaOH was added and the cells were incubated for an additional 4h. Cells were then harvested by centrifugation (10000g, 10min) and frozen at -20degC. |
| Method of Induction | Nalidixic Acid |
| Cell Density at Induction | OD 600 = 2.5 |
| 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 | 4.5M urea, 10mM Tris |
| Refolding Buffer | 10mM TrisHCl pH 9.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 64h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | The cells were then thawed and resuspended 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 washed twice with 1% Triton X-100 followed by two washing with water. The inclusion body pellet was then solubilized in solubilization buffer, the pH was then adjusted to 11.3 with NaOH and cysteine was added to 1mM. The solution was gently stirred at 4degC for 6h, diluted with 3 volumes of 0.67M L-arginine, stirred for an additional 10h, then dialyzed for 48h against 5x 10L refolding buffer. The protein was then purified further using a Q-sepharose column |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.67M |
| Refolding Yield | n/a |
| Purity | 90% |
| Notes | n/a |