Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lutropin Receptor |
| Abbreviated Name | LHR |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | P16235 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 711 |
| Molecular Weight | 78036.0 |
| Pi | 8.53291 |
| Molecular Weight | 78036.0 |
| Disulphides | Unknown |
| Full Sequence |
MGRRVPALRQLLVLAVLLLKPSQLQSRELSGSRCPEPCDCAPDGALRCPGPRAGLARLSL
TYLPVKVIPSQAFRGLNEVVKIEISQSDSLERIEANAFDNLLNLSELLIQNTKNLLYIEP
GAFTNLPRLKYLSICNTGIRTLPDVTKISSSEFNFILEICDNLHITTIPGNAFQGMNNES
VTLKLYGNGFEEVQSHAFNGTTLISLELKENIYLEKMHSGAFQGATGPSILDISSTKLQA
LPSHGLESIQTLIALSSYSLKTLPSKEKFTSLLVATLTYPSHCCAFRNLPKKEQNFSFSI
FENFSKQCESTVRKADNETLYSAIFEENELSGWDYDYGFCSPKTLQCAPEPDAFNPCEDI
MGYAFLRVLIWLINILAIFGNLTVLFVLLTSRYKLTVPRFLMCNLSFADFCMGLYLLLIA
SVDSQTKGQYYNHAIDWQTGSGCGAAGFFTVFASELSVYTLTVITLERWHTITYAVQLDQ
KLRLRHAIPIMLGGWLFSTLIATMPLVGISNYMKVSICLPMDVESTLSQVYILSILILNV
VAFVVICACYIRIYFAVQNPELTAPNKDTKIAKKMAILIFTDFTCMAPISFFAISAAFKV
PLITVTNSKILLVLFYPVNSCANPFLYAIFTKAFQRDFLLLLSRFGCCKRRAELYRRKEF
SAYTSNCKNGFPGASKPSQATLKLSTVHCQQPIPPRALTH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chen W, Bahl OP. (1993) Mol Cell Endocrinol, 91, 35-41 |
| 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 | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pT7-7-LHR(1-294) |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | unknown |
| Solubilization Buffer | 6M guanidine-HCl in the presence of 50mM DTT |
| Refolding Buffer | 1.5 M guanidinium chloride, 125mM Tris-HCl, 1mM cystine, 1mM cysteine |
| Pre-Refolding Purification | Ion-exchange + size exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 12-16h |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The expressed protein was centrifuged followed by solubilization. The expressed protein was centrifuged again followed by pre-purification, by size-exclusion chromatography. The fractions obtained were pooled and precipated by dialysis and solubilised in 8M urea. The solubilized protein was pre-purified by ion exchange chromatography. The purified protein was diluted with the unfolding buffer to 40ml. 20 ml of 1M Tris pH 7.5 was added and the solution was further diluted to 160ml with water and the redox agent. The solution was kept overnight at 4 degrees and then dialyzed against 0.1% NH4HCO3. The dialyzed solution was lyophilized, dissolved in 50mM Tris-HCl and centifuged. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | some contaminating bands on SDS |
| Notes | |