Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane protein A |
| Abbreviated Name | OmpA |
| SCOP Family | Outer membrane protein |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | Q9L6J0 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Transmembrane domain aa.1-171 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 171 |
| Molecular Weight | 18729.7 |
| Pi | 5.74705 |
| Molecular Weight | 18729.7 |
| Disulphides | 0 |
| Full Sequence |
APKDNTWYT GAKLGWSQYH DTGFINNNGP THENQLGAGA FGGYQVNPYV GFEMGYDWLG RMPYKGSVEN GAYKAQGVQL TAKLGYPITD DLDVYTRLGG MVWRADTKSN VYGKNHDTGV SPVFAGGVEY AITPEIATRL EYQWTNNIGD AHTIGTRPDN GMLSLGVSYR FG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Pautsch A, Vogt J, Model K, Siebold C, Schulz GE (1999) Proteins: Structure, Function, and Genetics, 34, 167-172 |
| Project Aim | Crystallography |
| 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 | 37.0 |
| Expression Time | 5h |
| Expression Vector | pET3b |
| Expression Protocol | Cells were grown in 250ml LB medium with 0.1mg/ml ampicillin at 37degC. Expression was induced when OD578 reached 0.8 with 1mM IPTG, cells were grown for a further 5h and harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 578 = 0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 20mM TrisHCl pH 8.5, 2%(w/v) TritonX-100 |
| Solubilization Buffer | 20mM TrisHCl pH 8.5, 6M Gdn |
| Refolding Buffer | 20mM TrisHCl pH 8.5, 5% (w/v) n-octylpolyoxyetheylene (C8POE), 0.6M arginine/20mM TrisHCl pH 8.5, 1% (w/v) C8POE |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 48h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet was resuspended in 30ml of 20mM Tris pH 8.5, then sonicated at 4degC. The inclusion bodies were centrifuged (90min x 4300g) then resuspended in wash buffer and centrifuged again. The inclusion bodies were then dissolved in 30ml solubilization buffer and incubated for 2h at 37degC (200rpm). The protein was then diluted into 150ml refolding buffer with 5%(w/v) C8POE, and dialyzed twice for 24h against 1L refolding buffer with 1% C8POE. The protein was then loaded onto a Source-Q ion exchange column incubated with 0.6%(w/v) C8P4 in 20mM Tris pH 8.5, and washed with 140ml of the same buffer. Protein was eluted by a 0-150mM NaCl gradient. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.6M |
| Refolding Yield | |
| Purity | |
| Notes | Protocol almost identical to that of OmpX, record no.1494 |