Refolding Record:
| Protein | |
|---|---|
| Protein Name | Olfactory receptor 5 |
| Abbreviated Name | OR5 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bombyx mori |
| UniProt Accession | Q4W1W3 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 677 |
| Molecular Weight | 79315.6 |
| Pi | 8.51 |
| Molecular Weight | 79315.6 |
| Disulphides | Unknown |
| Full Sequence |
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPVEMSHHHHHHGEFRESSISHAVPLEEAWDPSTNSS HPNTQV EEKVNNVEEF TYMKFLKSFC KIMDFWPERE EKNSKTRIFR LRYILVLQFC FTLVAGVLYL KNNFGKKTFY DLGHTIITVV MNVVSVSRLI LRCFKKYDVV GQQFINKIHL YHFRNDSEYS MKTYKAVHKI SNNMTYIFSF SIFVCVVTFN LNPVFNNIGS GAYKNPRPDN VTLQQCVYYA LPFDYTGDFK WYMLVAIFNV QKTFFCTSLF ILFDLLLSMM IIHLWGHIRI FIHNLNHIPA PRNSLEYTRE ERQEVDNTLK KCIQHHTLII GFVRIMSETY GLAVLIYYAF QQVVGCLLLL QCSRLDLKTI TRFGFLTTMV NQQLIQISVI FELLGYMNDK LQEAVYCVPW EYMDTSHRKM VYMMFRQSQI PLQLKAMNML SIGVKTMASI LKTSVTYYLM LKTITANEA
|
| Notes | GST-OR5 construct (basically full length protein) |
| Expression | |
|---|---|
| Report | Kiefer H, Krieger J, Olszewski JD, von Heijne G, Prestwhich GD, Breer H (1996) Biochemistry, 35, 16077-16084 |
| Project Aim | Functional Studies |
| Fusion | N-terminal GST and C-terminal hexahis |
| 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 | 4h |
| Expression Vector | pGEX-2C |
| Expression Protocol | 100ml of LB-ampicillin (100microg/ml) was inoculated with 2ml overnight culture and grwon at 37degC |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20mM Tris pH 7.5, 150mM NaCl |
| Solubilization Buffer | 20mM Tris pH 7.5, 150mM NaCl, 1.25(w/v) N-lauroylsarcoine |
| Refolding Buffer | 50mM Tris pH 8.5, 0.2mM DTT |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 4h |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.2mM |
| Refolding Protocol | Resuspended cells in 10mM Tris, 25% sucrose pH 7.5 had 1mM EDTA and 0.25mg of lysozyme added and cells were then stirred on ice for 1h. 5mM DTT and 200microM PMSF were added and cells were lysed by sonication. Insoluble material was collected by centrifugation (150000g, 2h). The pellet was resuspended in 20ml wash buffer and then re-centrifuged in the same manner. The pellet was then resuspended in 1ml of TBS (20mM Tris pH 7.5, 150mM NaCl), 1mM DTT and stored at -20degC. For solubilization, 4ml of solubilization buffer was added and the mixture was sonicated in a bath sonicator for 1min at 0degC. 20ml of TBS with 0.2% digitonin was added and the sample was centrifuged (20000g, 10min). The supernatant was loaded onto a Ni-NTA agarose column and the protein was eluted in TBS/0.1% digitonin with a gradient of 0-0.3M imidzaole. Selected fractions were pooled and dialysed against refolding buffer for 4h. 10units of thrombin and 0.5% sarcosyl were added and the sample was incubated for 15h at 20degC. The protein was diluted to 25ml with TBS, 0.2% digitonin, 0.5% sarcosyl, then rechromatographed on a Ni-NTA column. |
| Refolding Assay | Far-UV Circular Dichroism,Fluorescence,Ligand Binding,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |