Refolding Record:
| Protein | |
|---|---|
| Protein Name | Rad51B Protein |
| Abbreviated Name | Rad51B |
| SCOP Family | Helicase |
| Structure Notes | |
| Organism | Physcomitrella patens |
| UniProt Accession | Q93W51 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 342 |
| Molecular Weight | 36857.9 |
| Pi | 5.71 |
| Molecular Weight | 36857.9 |
| Disulphides | Unknown |
| Full Sequence |
MATASAATEGATTSAATEEEIHHGPYLVEHLQSCGISALDLKKLKDAGYCTVEAVAYSAK
KDLVNIKGLSDAKVDKIIEAAGKLVPMGFTSAKQMHEQRAELIQITTGSKEFDSILEGGI
ETGSITEIYGEFRSGKSQICHTLCVTCQLPLDQGGGEGKALYIDAEGTFRPQRLLQIAEK
YGLNGQDVLDNVAYARAYNTDHQTKLLVEAASMMAETRFALMVVDSATALYRTDYSGRGE
LAARQFHLAKFLRGCQKMADEFGIAVVVTNQVVAQVDGSAMFNGPQFKPIGGNIIAHAST
TRLSVRKGRGEERVIKVVASPCLAEQEARFQITNEGVVDVKE
|
| Notes | Sequence provided refers to recA protein only. |
| Expression | |
|---|---|
| Report | Ayora, S., Piruat, J. I., Luna, R., Reiss, B., Russo, V. E. A., Aguilera, A., Alonso, J. C. (2002) J Mol Biol, 316, 35-49 |
| Project Aim | Undefined |
| Fusion | None |
| 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 | 2.5 |
| Expression Vector | pET-3b |
| Expression Protocol | 30 min after induction with IPTG, 150 microg/ml rifampicin was added and cells were incubated for a |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 560 = 0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | not stated |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM TrisHCl, pH 7.5, 1 M NaCl, 1 mM DTT, 0.5 mM EDTA, 5%(v/v) glycerol |
| Solubilization Buffer | 4 M urea, 50 mM TrisHCl, pH 7.5, 1 mM DTT, 0.5 mM EDTA, 5%(v/v) glycerol |
| Refolding Buffer | 500 mM NaCl, 50 mM TrisHCl, pH 7.5, 1 mM DTT, 0.5 mM EDTA, 5%(v/v) glycerol |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | Inclusion bodies were washed three times in washing buffer, and then solubilized. The solution was then centrifuged and the supernatant loaded onto a Q-Sepharose column equilibrated with solubilization buffer containing 10 mM NaCl. Proteins were then eluted by using a linear gradient from 10-100 mM NaCl and 4 M urea. Rad51 were refolded by dialysis against equal volumes of refolding buffer. They were then dialyzed against refolding buffer containing 300 mM NaCl, 50% glycerol rather than 500mM Nacl and 5% glycerol. This solution was then centrifuged(45,000rpm, 1h) and stored at -20degC. |
| Refolding Assay | DNA binding |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 5% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |