Refolding Record:
| Protein | |
|---|---|
| Protein Name | Glutamyl-tRNA reductase |
| Abbreviated Name | GluTR |
| SCOP Family | Glutamyl tRNA-reductase catalytic, N-terminal domain |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | Q8FI03 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Tetramer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 442 |
| Molecular Weight | 48873.6 |
| Pi | 5.76478 |
| Molecular Weight | 48873.6 |
| Disulphides | 0 |
| Full Sequence |
MGSSHHHHHHSSGLVPRGSHMLGD MTLLALGINH KTAPVSLRER VSFSPDKLDQ ALDSLLAQPM VQGGVVLSTC NRTELYLSVE ERDDLQEALI RWLCDYHNLN EDDLRNSLYW HQDNDAVSHL MRVASGLDSL VLGEPQILGQ VKKAFADSQK GHMKASELER MFQKSFSVAK RVRTETDIGA SAVSVAFAAC TLARQIFESL STVTVLLVGA GETIELVARH LREHKVQKMI IANRTRERAQ ILADEVGAEV IALSDIDERL READIIISST ASPLPIIGKG MVERALKSRR NQPMLLVDIA VPRDVEPEVG KLANAYLYSV DDLQSIISHN LAQRKAAAVE AETIVAQEAS EFMAWLRAQS ASETIRDYRS QAEQVRDELT AKALAALEQG GDAQTIMQDL AWKLTNRLIH APTKSLQQAA RDGDNERLNI LRDSLGLE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Schauer S, Luer C, Moser J (2003) Protein Expression and Purification, 31, 271-275 |
| Project Aim | Biophysical 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 | 2h |
| Expression Vector | pET15b |
| Expression Protocol | Cells were grown in 150 ml Luria-Bertani medium containing 100 microg/ml ampicillin at 37degC at 250 rpm for 15 h from a single colony. Four one-liter Erlenmeyer flasks, each containing 500 ml LB medium with 100 microg/ml ampicillin, were inoculated with 5 ml of an overnight culture and the cells were cultivated at 37degC with vigorous shaking (250 rpm) to a cell density of 0.7 A578. After the addition of IPTG to a final concentration of 250microM and 50microg ampicillin to every flask, cultures were further incubated at 37degC for 2 h to an A578 of 2.5. Subsequently, cells were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 578 = 0.7 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM Tris-HCl, 6M guanidinium chloride, 0.5M NaCl, 5mM betamercaptoethanol, pH 8 |
| Refolding Buffer | 20mM Tris-HCl, 20% glycerol, 0.5M NaCl, 5mM betamercaptoethanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 20h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 5mM,5mM |
| Refolding Protocol | The inclusion bodies were solubilized in 200 times their weight of 20 mM Tris (HCl), pH 8.0, containing 6 M guanidineHCl, 500 mM NaCl, and 5 mM beta-mercaptoethanol (buffer S), centrifuged for 15 min at 47,800g (Sorvall SS 34 rotor, 20,000 rpm) at 4degC, and applied to 40 ml Ni2+-loaded Chelating Sepharose Fast Flow (Amersham, Freiburg, Germany) in a XK 26/20 column (Amersham, Freiburg, Germany) pre-equilibrated with buffer S at a flow rate of 6 ml/min. After binding the buffer was changed to 20 mM Tris (HCl), pH 8.0, including 6 M urea, 500 mM NaCl, and 5 mM beta-mercaptoethanol (buffer F) and a linear gradient over 30 column volumes at 1.2 ml/min at room temperature to 20 mM Tris (HCl), pH 8.0, with 20% (v/v) glycerol, 500 mM NaCl, and 5 mM beta-mercaptoethanol (buffer W) was performed. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | 9mg/L culture; approx. 10% (w/w) of inclusion body |
| Purity | |
| Notes | |