Refolding Record:
| Protein | |
|---|---|
| Protein Name | Leptin |
| Abbreviated Name | Leptin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q28603 |
| 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 | 167 |
| Molecular Weight | 18348.2 |
| Pi | 7.92 |
| Molecular Weight | 18348.2 |
| Disulphides | 1 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHM VPIRKVQDDT KTLIKTIVTR INDISHTQSV SSKQRVTGLD FIPGLHPLLS LSKMDQTLAI YQQILASLPS RNVIQISNDL ENLRDLLHLL AASKSCPLPQ VRALESLESL GVVLEASLYS TEVVALSRLQ GSLQDMLRQL DLSPGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Varnerin JP, Smith T, Rosenblum CI, Vongs A, Murphy BA, Nunes C, Mellin TN, King JJ, Burgess BW, Junker B, Chou M, Hey P, Frazier E, MacIntyre DE, Van der Ploeg LH, Tota MR (1998) Protein Expression and Purification, 14, 335-342 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HMS174(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | pET27b |
| Expression Protocol | The protein was expressed in the E.coli strain HMS174(DE3) pLysS (Novagen) by growing the cells at 37degC in LB medium with kanamycin (30 mg/ml) to an OD600 of 0.6 followed by induction with 1 mM IPTG. After 2h further incubation, the cells were chilled at 15degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Diafiltration |
| Wash Buffer | 50mM Tris-HCl, 0.5% Triton X-100, pH 8.0 |
| Solubilization Buffer | 50mM Tris-HCl, 6M guanidinium chloride, pH 8.0 |
| Refolding Buffer | 20mM Tris-HCl |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 55h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 5mM/0.5mM |
| Refolding Protocol | Large scale refolding was achieved by utilizing a 10-KDa MWCO spiral wound membrane cartridge. Three grams of metal affinity purified material was diluted to 1.5mg.ml in 6M guanidinium chloride, 20mM Tris-HCl, 2.5mM beta mercaptoethanol, 2.5mM DTT, pH 8.0. Over 30 min this material was diluted 10-fold into 6M urea, 20mM Tris-HCl, 5mM beta mercaptoethanol, pH 7.4. Diafiltration was performed in 3 steps: 1 vol of 20mM Tris-HCl, 5mM beta mercaptoethanol, pH 7.4 for 20h; 1 vol of Tris-HCl, 1mM DTT, pH 7.4 for 5h; and 2 vol of of argon purged 20mM Tris-HCl pH 7.4 for 18h. 5mM GSSG and 0.5mM GSH were added and incubated overnight. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 52.9% |
| Purity | 95% |
| Notes | |