Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane protein F |
| Abbreviated Name | OmpF |
| SCOP Family | Porin |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P02931 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 341 |
| Molecular Weight | 37215.7 |
| Pi | 4.64 |
| Molecular Weight | 37215.7 |
| Disulphides | 0 |
| Full Sequence |
MAEIYNKDG NKVDLYGKAV GLHYFSKGNG ENSYGGNGDM TYARLGFKGE TQINSDLTGY GQWEYNFQGN NSEGADAQTG NKTRLAFAGL KYADVGSFDY GRNYGVVYDA LGYTDMLPEF GGDTAYSDDF FVGRVGGVAT YRNSNFFGLV DGLNFAVQYL GKNERDTARR SNGDGVGGSI SYEYEGFGIV GAYGAADRTN LQEAQPLGNG KKAEQWATGL KYDANNIYLA ANYGETRNAT PITNKFTNTS GFANKTQDVL LVAQYQFDFG LRPSIAYTKS KAKDVEGIGD VDLVNYFEVG ATYYFNKNMS TYVDYIINQI DSDNKLGVGS DDTVAVGIVY QF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Conlan S, Zhang Y, Cheley S, Bayley H (2000) Biochemistry, 39, 11845-11854 |
| 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 | PC2889E |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | pT7-SMC |
| Expression Protocol | 400ml LB medium with 50microg/ml ampicillin was inoculated with 5ml overnight culture. Cells were grown until A600 reached 0.7, expression was then induced with 0.5mM IPTG and grown for a further 2h. The cells were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 1M urea, 1% Triton X-100, 10mM TrisHCl pH 8.0 |
| Solubilization Buffer | 8M urea, 10mM TrisHCl pH 8.0 |
| Refolding Buffer | 1% (w/v) Genapol X-080, 10mM TrisHCl pH 8.0 |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were lysed in 5ml of 200microg/ml lysozyme, 1mM PMSF, 10mm TrisHCl, 1mM EDTA pH 8.0 on ice for 15min. The lysate was centrifuged (60000g) and the pellet was washed once in 10ml wash buffer containing Triton X-100, and once without Triton X-100. The pellet was then dissolved at 4degC in 50ml solubilization buffer. The protein was filitered and loaded onto a DEAE-sepharose column. The protein was eluted with a linear gradient of 0-500mM (150ml) NaCl in solubilization buffer. Selected fractions were pooled and dialyzed against 2L of refolding buffer with one buffer change at 4degC. |
| Refolding Assay | Far-UV Circular Dichroism,Bioactivity,SDS-PAGE,Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 12.5mg/L |
| Purity | n/a |
| Notes | n/a |