Refolding Record:
| Protein | |
|---|---|
| Protein Name | Beta-2-microglobulin |
| Abbreviated Name | Beta2m |
| SCOP Family | C1 set domains (antibody constant domain-like) |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P61769 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 99 |
| Molecular Weight | 11731.2 |
| Pi | 6.07 |
| Molecular Weight | 11731.2 |
| Disulphides | 1 |
| Full Sequence |
IQRTPKIQVY SRHPAENGKS NFLNCYVSGF HPSDIEVDLL KNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Garboczi DN, Hung DT, Wiley CD (1992) Proc. Natl. Acad. Sci. USA, 89, 3429-3433 |
| Project Aim | Crystallography |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XA90 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pHN1+ |
| Expression Protocol | 1L of cells was incubated at 37degC and induced by the addition of 1mM IPTG. When OD650 reached 1.8-2.0, cells were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 100mM TrisHCl pH 8.0m 8M urea |
| Refolding Buffer | 10mM TrisHCl pH 7.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were resuspended in 20ml of 10mM TrisHCl pH 8 containing 100microg/ml lysozyme, 50microg/ml PMSF, 20microg/ml DNase, 20microg/ml RNase and 1mM EDTA. Cells were incubated for 20min at 22degC, then lysed by conication and centrifuged (20min, 10000g). The pellet was washed in 20ml wash buffer, then dissolved in 10ml solubilization buffer and centrifuged (150000, 1h, 4degC). The protein was then refolded by dialysis against refolding buffer and purified on Q sepharose in 10mM TrisHCl pH 7 with a linear gradient from 0-100mM NACl. Selected fractions werer dialyzed against water and concentrated |
| Refolding Assay | Ultraviolet (UV) Absorbance |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |