Refolding Record:
| Protein | |
|---|---|
| Protein Name | Beta-lactoglobulin A |
| Abbreviated Name | BLGA |
| SCOP Family | Retinol binding protein-like |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P02754 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 162 |
| Molecular Weight | 18367.3 |
| Pi | 4.75979 |
| Molecular Weight | 18367.3 |
| Disulphides | 2 |
| Full Sequence |
LIVT QTMKGLDIQK VAGTWYSLAM AASDISLLDA QSAPLRVYVE ELKPTPEGDL EILLQKWEND ECAQKKIIAE KTKIPAVFKI DALNENKVLV LDTDYKKYLL FCMENSAEPE QSLVCQCLVR TPEVDDEALE KFDKALKALP MHIRLSFNPT QLEEQCHI
|
| Notes | mutants K70M, F136A and K141M also made |
| Expression | |
|---|---|
| Report | Cho Y, Batt CA, Sawyer L (1994) J Biol Chem, 269, 11102-11107 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | JM105 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pTTQ18 |
| Expression Protocol | Cells were grown in LB medium with 50microg/ml at 37degC and induced with 0.5mM IPTG when A600 reached 0.5. Cells were then incubated for 4h |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | distilled water |
| Solubilization Buffer | 0.1M TrisHCl pH 8, 8M urea, 2mM DTT |
| Refolding Buffer | 0.1M TrisHCl pH 8, 1mM GSSG, 0.5mM GSH, 0.2mM EDTA |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 22.0 |
| Protein Concentration | n/a |
| Refolding Time | 12h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 0.5/1mM,0.5/1mM,0.5/1mM |
| Refolding Protocol | Cells were resuspended in 50mM TrisHCl pH 8 and sonicated. After centrifugation (12000g, 20min), the insoluble pellet was washed once with distilled water, then dissolved in solubilization buffer. After centrifugation, the supernatant was diluted 20-fold with refolding buffer and incubated at 0degC for 10min. The temperature was then raised to 22degC and the solution was stirred for 12h. The pH of the solution was adjusted to 2.8 with HCl and the resulting precipitate was removed by centrifugation. The protein was then purified by gel filtration using a Sephadex G-50 column. |
| Refolding Assay | Fluorescence,Ligand Binding,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |