Refolding Record:
| Protein | |
|---|---|
| Protein Name | RNA polymerase |
| Abbreviated Name | RNAP |
| SCOP Family | RNA-polymerase beta-prime |
| Structure Notes | |
| Organism | Bordetella pertussis |
| UniProt Accession | P0A4E5 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Multi-domain proteins (alpha and beta) |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Alpha Chain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 328 |
| Molecular Weight | 36158.5 |
| Pi | 5.6 |
| Molecular Weight | 36158.5 |
| Disulphides | 0 |
| Full Sequence |
MSTQGFLKPRSIEVEPVGAHHAKIVMEPFERGYGHTLGNALRRILLSSMTGYAPTEVQMT
GVVHEYSTIAGVREDVVDILLNLKGVVFKLHNRDEVTLVLRKNGAGAVVASDIELPHDVE
IINPDHLICNLTDAGKIEMQVKVEKGRGYVPGNVRALSEDRTHTIGRIVLDASFSPVRRV
SYAVESARVEQRTDLDKLVLDIETNGVISPEEAVRQAARILMDQISVFAALEGAGDAYEP
PVRGTPQIDPVLLRPVDDLELTVRSANCLKAENIYYIGDLIQRTENELLKTPNLGRKSLN
EIKEVLAARGLTLGMKLENWPPLGLERP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Boucher, P. E., Maris, A. E., Yang, M. S., Stibitz, S. (2003) Mol Cell, 11, 163-173 |
| Project Aim | Protein-Protein Interaction Identification |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M GuHCl, 50 mM TrisHCl, pH 7.9, 10 mM MgCl, 10 microM ZnCl2, 1 mM EDTA, 10% glycerol |
| Refolding Buffer | 50 mM Tris, pH 7.9, 0.2 M KCl, 10 mM MgCl2, 0.1 mM EDTA, 10 microM ZnCl2, 5 mM beta-mercaptoethanol, 10% glycerol |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.9 |
| Refolding Temperature | 4.0 |
| Protein Concentration | overnight |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 5mM |
| Refolding Protocol | Protein was isolated using Hi-Trap chelating column. Fractions containing alpha subunit were dialyzed against conjugation buffer (20mM MOPS-NAOH,pH 8.0, 10 mM MgCl2, 0.2 M KCl, 0.1 mM EDTA, 6 M urea. After incubation for 1h at 37degC, the rection was quenched by the addition of an equal volume of 1 M TrisHCl, pH 8.0. The sample was then passed through a PD10 column equilibrated with solubilization buffer. Protein was refolded by mixing in 10ml dissociation buffer containing 10mM DTT for 30min and then centrifuging (30min, 10,000g). The supernatant was then dialyzed overnight at 4degC against refolding buffer, with two changed of buffer. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |