Refolding Record:
| Protein | |
|---|---|
| Protein Name | PR domain-containing protein 2 Also known as Zinc finger protein RIZ |
| Abbreviated Name | RIZ1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q13029 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | RIZ-PR-A |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 321 |
| Molecular Weight | 36378.9 |
| Pi | 4.36 |
| Molecular Weight | 36378.9 |
| Disulphides | Unknown |
| Full Sequence |
MNQNTTEPVAATETLAEVPEHVLRGLPEEVRLFPSAVDKTRIGVWATKPILKGKKFGPFVGDKKKRSQVKNNVYMWEVYYPNLGWMCIDATDPEKGNWLRYVNWACSGEEQNLFPLEINRAIYYKTLKPIAPGEELLVWYNGEDNPEIAAIEEERASARSKRSSPKSRKGKKKSQENKNKGNKIQDIQLKTSEPDFTSANMRDSAEGPKEDEEKPSASALEQPATLQEVASQEVPPELATPAPAWEPQPEPDERLEAAACEVNDLGEEEEEEEEEDEEEEEDDDDDELEDEGEEEASMPNENSVKEPEIRCDEKPEDLLEE
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Derunes C, Briknarova K, Geng L, Li S, Gessner CR, Hewitt K, Wu S, Huang S, Woods VI Jr, Ely KR. (2005) Biochemical and Biophysical Research Com, 333, 925-934 |
| Project Aim | Expression and charactrization |
| Fusion | 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 | 4-5 h |
| Expression Vector | pKGRIZ322 |
| Expression Protocol | Plasmids encoding the proteins for study as glutathione-S-transferase (GST)-fusion proteins were transformed into Escherichia coli (E. coli) BL21(DE3) competent cells (Stratagene). For protein expression, a 300 ml overnight culture was diluted into 3 liters of LB medium containing 100 μg/ml ampicillin and grown with shaking at 37 °C to a cell density of A600 = 0.6. The temperature was decreased to room temperature and protein expression was induced by the addition of 0.5–0.8 mM isopropyl-1-thio-β-d-galactopyranoside (IPTG). The cultures were incubated for 4–5 h at room temperature. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea, 2 mM PMSF, 1 mM EDTA, and 5 mM β-mercaptoethanol, |
| Refolding Buffer | 20 mM potassium phosphate, pH 6.8, 50 mM NaCl, 1 mM PMSF, and 5 mM β-mercaptoethanol |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | yes |
| Refolding pH | 6.8 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | Renaturation of RIZ-PR-A Purified RIZ-PR-A protein was denatured and refolded prior to crystallization experiments and deuterium exchange experiments. Denaturation was achieved by dialysis at room temperature versus 8 M urea, 2 mM PMSF, 1 mM EDTA, and 5 mM β-mercaptoethanol, after which the protein was refolded by stepwise dialysis versus decreasing concentrations of urea. Finally, all urea was removed by dialysis against 20 mM potassium phosphate, pH 6.8, 50 mM NaCl, 1 mM PMSF, and 5 mM β-mercaptoethanol. The last purification step of renatured RIZ-PR-A involved ion-exchange chromatography on a Q-Sepharose column equilibrated with the last dialysis buffer. Bound protein was eluted from the column using a salt gradient (50–600 mM NaCl). Purified protein was stored at 4 °C in 10 mM glycine, pH 7.0, 50 mM NaCl, 1 mM PMSF, and 5 mM β-mercaptoethanol. |
| Refolding Assay | Mass spectrometry,NMR analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |