Refolding Record:
| Protein | |
|---|---|
| Protein Name | Toxin yoeB |
| Abbreviated Name | yoeB |
| SCOP Family | YoeB/Txe-like |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P69348 |
| 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 | 84 |
| Molecular Weight | 10215.6 |
| Pi | 7.86 |
| Molecular Weight | 10215.6 |
| Disulphides | Unknown |
| Full Sequence |
MKLIWSEESWDDYLYWQETDKRIVKKINELIKDTRRTPFEGKGKPEPLKHNLSGFWSRRITEEHRLVYAVTDDSLLIAACRYHY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kamada K, Hanaoka F. (2005) Mol Cell, 19, 497-509 |
| 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 | 0.0 |
| Expression Time | 00 |
| Expression Vector | pET11a |
| Expression Protocol | DNA fragments encoding full-length yefM and yoeB were amplified from E. coli K-12 genomic DNA by PCR and subcloned under the T7 promoter in pET-28a and pET-11a vectors (Novagen), respectively. Full-length and an N-terminal truncated form (amino acids 10–92) of YefM were expressed with an N-terminal hexa-histidine tag and a human rhinovirus 3C protease cleavage site. YoeB was expressed without any tags. These two vectors were cotransformed into E. coli BL21(DE3) to produce the antitoxin-toxin complex. Following cell lysis, the oligomeric YefM-YoeB complex was purified via Ni2+ affinity chromatography, and the hexa-histidine tag was removed by proteolysis. Serial column chromatography on heparin-Sepharose and Q-Sepharose yielded highly purified toxin-antitoxin complexes. Selenomethionine-labeled (Se-Met) YefM-YoeB complexes were expressed and purified using the same procedure as described above. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M Guanidine-HCl |
| Refolding Buffer | 25 mM HEPES (pH 7.5), 200 mM NaCl, and 1 mM DTT at 4°C |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | YefM-free YoeB was extracted from a Ni2+ chelating column, on which the YefM-YoeB heterocomplex had been immobilized via the histidine-tagged YefM, by a buffer containing 6M Guanidine-HCl. The fraction of denatured YoeB was adjusted to 0.5 mg/ml and then refolded by dialyzing gradually against 25 mM HEPES (pH 7.5), 200 mM NaCl, and 1 mM DTT at 4°C. The refolded YoeB was further purified to homogeneity by Heparin-Sepharose and gel filtration columns. The hexa-histidine tagged YefM antitoxin (amino acids 10–92) was expressed alone and purified via Ni2+ affinity chromatography. After removal of the hexa-histidine tag, YefM was further purified using a Q-Sepharose column. |
| Refolding Assay | Analytical centrifugation,GST Pull-Down Assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |