Refolding Record:
| Protein | |
|---|---|
| Protein Name | Growth Hormone Receptor |
| Abbreviated Name | GHR |
| SCOP Family | Fibronectin type III |
| Structure Notes | |
| Organism | Ovis aries (sheep/ovine) |
| UniProt Accession | Q28575 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 626 |
| Molecular Weight | 68958.4 |
| Pi | 4.56144 |
| Molecular Weight | 68958.4 |
| Disulphides | 3 |
| Full Sequence |
FSGSEATPAFFVRASQSLQILYPGLETNSSGNLKFTKCRSPE
LETFSCHWTDGANHSLQSPGSVQMFYIRRDIQEWKECPDYVSAGENSCYFNSSYTSVWTP
YCIKLTSNGGIVDHKCFSVEDIVQPDPPVGLNWTLLNISLTEIHADILVKWEPPPNTDVK
MGWIILEYELHYKELNETQWKMMDPLLVTSVPMYSLRLDKEYEVRVRTRQRNTEKYGKFS
EVLLITFPQMNPSACEEDFQFPWFLIIIFGILGLTVTLFLLIFSKQQRIKMLILPPVPVP
KIKGIDPDLLKEGKLEEVNTILAIHDNYKHEFYNDDSWVEFIELDIDDPDEKTEGSDTDR
LLSNDHEKSLSIFGAKDDDSGRTSCYEPDILETDFHVSDMCDGTSEVAQPQRLKGEADIL
CLDQKNQNNSPSNDAAPASQQPSVILVEENKPRPLPIGGTESTHQAVHTQLSNPSSLANI
DFYAQVSDITPAGNVVLSPGQKNKTGNPQCDTHPEVVTPSQADFIVDSAYFCEVDAKKYI
ALAPDVEAESHIEPSFNQEDIYITTESLTTTAGRSGTAENVPSSEIPVPDYTSIHIVQSP
QGLVLNATALPLPDKEFLSSCGYVSTDQLNKIMP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Allan, G. J., Shand, J. H., Beattie, J., and Flint, D. J. (1999) Eur J Biochem., 261, 555-561 |
| Project Aim | Structure-Function |
| 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(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 2-2.5h |
| Expression Vector | pET15b |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 1 mM EDTA, 4.5 mM Urea, 50 mM Tris, pH 9.5 |
| Refolding Buffer | 20 mM TrisHCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 20h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | n/a |
| Refolding Protocol | 1 L of LB medium containing ampicillin and chloramphenicol was inoculated with an overnight culture and allowed to grow until the OD600 reached approximately 0.6. IPTG was then added to a final concentration of 1 mM. Cells were harvested by centrifugation (17000g, 15 min) and washed. The pellet was then resuspended in NaCl//P1 containing a protease inhibitor cocktail to a total volume of 10 ml and frozen at -70degC. Pellet was then thawed and 100 microml of 10 %(v/v) Triton X-100 was added before the suspension was sonicated 3x 30 seconds. The lysate was centrifuged (13 000g, 15 min) and supernatant discarded before the addition of solubilization buffer. Refolding occurred through a combination of dilution and dialysis against three changes of refolding buffer. The refolded protein was purified by collection in a disposable column before elution using imidazole. The purity of the protein was later assessed by SDS page and Coomassie staining. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | Extracellular Domain AA 1-267 Expressed and Refolded MGSSHHHHHHSSGLVPRGSH MAFS GSEATPAFFV RASQSLQILY PGLETNSSGN 50 LKFTKCRSPE LETFSCHWTD GANHSLQSPG SVQMFYIRRD IQEWKECPDY 100 VSAGENSCYF NSSYTSVWTP YCIKLTSNGG IVDHKCFSVE DIVQPDPPVG 150 LNWTLLNISL TEIHADILVK WEPPPNTDVK MGWIILEYEL HYKELNETQW 200 KMMDPLLVTS VPMYSLRLDK EYEVRVRTRQ RNTEKYGKFS EVLLITFPQM 250 NPSACEEDFQ FPW Number of amino acids: 267 Molecular weight: 30689.6 Theoretical pI: 5.71 |