Refolding Record:
| Protein | |
|---|---|
| Protein Name | Mu-type opioid receptor |
| Abbreviated Name | MOR-1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P35372 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Topological domain polypeptide containing residues 97–176 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 79 |
| Molecular Weight | 8960.6 |
| Pi | 8.53 |
| Molecular Weight | 8960.6 |
| Disulphides | Unknown |
| Full Sequence |
YTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kerman A, Ananthanarayanan VS. (2005) Biochemica et Biophysica Acta, 1747, 133-140 |
| Project Aim | Expression and charactrization |
| Fusion | N-terminal GST |
| 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 | 3-3.5 h |
| Expression Vector | pGX-2T |
| Expression Protocol | The plasmid pGEX-TM2–3, constructed as above, was transformed into the expression strain BL21(DE3). Overnight cultures were grown in LB containing 100 μg/ml ampicillin. Ten millilitres of overnight culture were then diluted into 1 L of LB containing 100 μg/ml ampicillin and grown at 37 °C with vigorous shaking (235 rpm) until the OD600 reached 0.6–0.8 (usually 2.5–3 h.) IPTG was added to a final concentration of 1 mM, and growth was continued for a further 3–3.5 h at 37 °C. Bacteria were then harvested by centrifugation at 4000×g for 10 min. Pellets were stored at −20 °C until used for purification. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-0.8 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | RP-HPLC |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mM Tris–HCl (pH 8.0), 2 M urea, 2% Triton X-100, 1 mM EDTA |
| Solubilization Buffer | 20 mM Tris–HCl (pH 8.0), 1.5% N-lauroylsarcosine , 100 mM β-mercaptoethanol |
| Refolding Buffer | tris-HC1 buffer |
| Pre-Refolding Purification | RP-HPLC |
| Tag Cleaved | yes |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 12 mM |
| Refolding Protocol | refolding? |
| Refolding Assay | Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 0.3% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | please do not submit this paper yet |