Refolding Record:
| Protein | |
|---|---|
| Protein Name | Leptin |
| Abbreviated Name | Leptin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | P50596 |
| 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 | 177 |
| Molecular Weight | 19547.5 |
| Pi | 5.81808 |
| Molecular Weight | 19547.5 |
| Disulphides | 1 |
| Full Sequence |
MKETAAAKFERQHMDSPDLGT LVPRGSMADI VPIHKVQDD TKTLIKTIVT RINDISHTQS VSARQRVTGL DFIPGLHPIL SLSKMDQTLA VYQQILTSLP SQNVLQIAHD LENLRDLLHL LAFSKSCSLP QTRGLQKPES LDGVLEASLY STEVVALSRL
QGSLQDILQQ LDLSPEC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Park J-H, Lee H-H, Na S-Y, Ju S-K, Lee Y-J, Lee M-K, Kim KL (2001) Protein Expression and Purification, 22, 60-69 |
| Project Aim | Functional Studies |
| Fusion | N-terminal S-tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET29 |
| Expression Protocol | Cells were grown and expression was induced in the log growth phase with 0.1mM IPTG. After 3h further incubation, cells were harvested by centrifugation (10min, 6000g) and the pellet was resuspended in 1/50 cell culture volume of wash buffer without Triton. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 1mM EDTA, 20mM NaCl, 25mM TrisHCl pH 7.4, 1mM fresh PMSF, 0.1% Triton X-100 |
| Solubilization Buffer | 8M urea, 25mM TrisHCl pH 7.4 |
| Refolding Buffer | 25mM TrisHCl pH 7.4 |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 60h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2.5mM/0.25mM |
| Refolding Protocol | Cell were disrupted by sonication and inclusion bodies were harvested by centrifugation (20min, 10000g). The pellet was then resuspended in 1/50 culture volume of wash buffer. This wash procedure was repeated once. The pellet was then washed once in distilled water then resuspended in solubilization buffer. The protein was refolded by stepwise dialysis into refolding buffer containing lower concentrations of urea, starting with 4M urea for 12 h, then by further 2-fold diluted urea buffer for every 12h. From 1M urea concentration, 2.5mM reduced glutathione and 0.25mM oxidized glutathione were added to the mixture. Stepwise dialysis was performed against progressively lower concentrations of urea until the urea concentration reached 0.25mM. The solution was then dialyzed against RPMI 1640 medium and filnnaly sterile filtered. The S-tag was removed by thrombin cleavage. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | 99% |
| Notes | |