Refolding Record:
| Protein | |
|---|---|
| Protein Name | RNA Dependent RNA Polymerase |
| Abbreviated Name | RdRp |
| SCOP Family | RNA dependent RNA polymerases |
| Structure Notes | |
| Organism | Hepititis E Virus |
| UniProt Accession | Q03495 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Multi-domain proteins (alpha and beta) |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 398 |
| Molecular Weight | 43613.3 |
| Pi | 6.67 |
| Molecular Weight | 43613.3 |
| Disulphides | 0 |
| Full Sequence |
M A A P S Q R K A V V S T L V G R Y G R R T K L Y N A S H S D V R D S L A R F I P A I G P V Q V T T C E L Y E L V E A M V E K G Q D G S A V L E L D L C N R D V S R I T F F Q K D C N K F T T G E T I A H G K V G Q G I S A W S K T F C A L F G P W F R A I E K A I L A L L P Q G V F Y G D A F D D T V F S A A V A A A K A S M V F E N D F S E F D S T Q N N F S L G L E C A I M E E C G M P Q G L I R L Y H L I R S A W I L Q A P K E S L L G F W K K H S G E P G T L L W N T V W N M A V I T H C Y D F R D L Q V A A F K G D D S I V L C S E Y R Q S P G A A V L I A G C G L K L K V D F R P M R L Y A G V V V A P G L G A L P D V V R F A G R L T E K N W G P G P E R A D E L R I A V S D F L R K L T N V A Q M C V D V V S R V Y G V S P G L V H N L I G M L Q A V A D G K A H F T E S V K P V L D L T N S I L C R V E
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Agrawal. S., Gupta, D., Panda, S. K. (2001) Virology, 282, 87-101 |
| 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 | 3h |
| Expression Vector | pRSET |
| Expression Protocol | Overnight culture grown in LB medium was inoculated at 1% into NZ-amine medium also containing 0.4% |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Chemical |
| Lytic Agent | None |
| Pre-Refolding Purification | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M urea, 500 mM NaCl, 20% glycerol, TrisHCl pH 7.4 containing phenylmethylsulfonyl fluoride. |
| Refolding Buffer | Reduced concentration of urea, 500 mM NaCl, 20% glycerol, TrisHCl pH 7.4 containing phenylmehtylsulfonyl fluoride |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | no |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Hexa-histidine tagged RdRP was prepurified on Ni2+- NTA-agarose before being immobilised on a column. Refolding was preformed over a linear 6- 1 M urea gradient in 500mM Nacl, 20% glycerol, TrisHCl ph 7.4 containing PMSF. Bound RdRp was eluted using 250 mM imidazol and checked on SDS 10% polyacrylamide gel. |
| Refolding Assay | RNA binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |