Refolding Record:
| Protein | |
|---|---|
| Protein Name | Rad52 |
| Abbreviated Name | Rad52 |
| SCOP Family | Homologous-pairing domain of Rad52 recombinase |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q8NG50 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 211 |
| Molecular Weight | 23287.3 |
| Pi | 8.16 |
| Molecular Weight | 23287.3 |
| Disulphides | Unknown |
| Full Sequence |
MSGTEEAILGGRDSHPAAGGGSVLCFGQCQYTAEEYQAIQKALRQRLGPEYISSRMAGGGQKVCYIEGHRVINLANEMFGYNGWAHSITQQNVDFVDLNNGKFYVGVCAFVRVQLKDGSYHEDVGYGVSEGLKSKALSLEKARKEAVTDGLKRALRSFGNALGNCILDKDYLRSLNKLPRQLPEVDLTKAKRQDLEPSVEEARYNSCRPNM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lloyd, JA., McGrew, DA & Knight, KL. (2005) J Mol Biol, 345, 239-249 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)CodonPlus |
| Expression Temp | 37.0 |
| Expression Time | Not stated |
| Expression Vector | Not stated |
| Expression Protocol | Transformed cultures of BL21 (DE3) Codon Plus were grown in 1/2×Superbroth (1.8 l) containing 50 μg/ml kanamycin and induced with 3 mM isopropyl-1-thio-β-D-galactopyranoside. Cells were resuspended in T-buffer (20 mM Tris (pH 7.9), 10% (w/v) glycerol) with 500 mM NaCl, 0.5% (w/v) NP-40, 5 mM imidazole. Protease inhibitors (1 mM phenyl methyl sulfonyl fluoride and 10 mM benzamidine) were used throughout the purification. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | not stated |
| Solubilization Buffer | 20 mM Tris (pH 7.9), 500 mM NaCl, and 7 M urea |
| Refolding Buffer | 20 mM Tris (pH 7.9), 200 mM (NH4)2SO4, and 10% glycerol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.9 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Native HsRad52(1-212) was unfolded by diluting purified protein with 20 mM Tris (pH 7.9), 500 mM NaCl, and 7 M urea to a final concentration of 6 M urea. The proteins were refolded by dialysis into 20 mM Tris (pH 7.9), 200 mM (NH4)2SO4, and 10% glycerol. Purified proteins were stored in 20 mM Tris (pH 7.9), 200 mM (NH4)2SO4, and 10% glycerol. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol,(NH4)2SO4 |
| Additives Concentration | 200 mM (NH4)2SO4, and 10% glyc |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |