Refolding Record:
| Protein | |
|---|---|
| Protein Name | Glucocorticoid-induced TNF receptor |
| Abbreviated Name | GITR |
| SCOP Family | TNF receptor-like |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | TNF18_MOUSE |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 167 |
| Molecular Weight | 18087.5 |
| Pi | 8.15458 |
| Molecular Weight | 18087.5 |
| Disulphides | 5 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHM QPSVVEEPGCG PGKVQNGSGN NTRCCSLYAP GKEDCPKERC ICVTPEYHCG DPQCKICKHY PCQPGQRVES QGDIVFGFRC VACAMGTFSA GRDGHCRLWT NCSQFGFLTM FPGNKTHNAV CIPEPLPTEQ YG RAPPPPPPLRSGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Shin HH, Lee MH, Kim SG, Lee YH, Kwon BS, Choi HS (2002) FEBS Letters, 514, 275-280 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 6h |
| Expression Vector | pET28a |
| Expression Protocol | Cells were grown at 37degC until OD600 reached 0.6-0.8. 0.4mM IPTG was added and the incubation continued at 37degC for a further 6h. The bacteria were then harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM TrisHCl pH 8.5, 1% SDS, 1mM 2-mercaptoethanol |
| Solubilization Buffer | 10mM Tris pH 8.0, 6M guanidine thiocyanate, 01% Triton X-100, 5mM 2-mercaptoethanol |
| Refolding Buffer | 10mM Tris pH 8.0, 5mM 2-mercaptoethanol |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 5mM,5mM |
| Refolding Protocol | The bacterial pellets wee washed twice in 20mM TrisHCl, pH 7.5, 10mM EDTA, 2% Triton X-100 and homogenized with a sonicator. The lysate was then dissolved in the same buffer, stirred for 30min at room temperature and centrifuged. The pellet was then washed three times and was stirred for 30min in wash buffer. The pellet was then dissolved in solubilization buffer and homogenized for 3min using a sonicator and centrifuged. The supernatant was dialyzed sequentially against refolding buffer with decreasing concentrations of guanidine thiocyanate (2.4, 0.5, 0.1, 0M) |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 14g/L culture |
| Purity | n/a |
| Notes | n/a |