Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane protein A |
| Abbreviated Name | OmpA |
| SCOP Family | Outer membrane protein |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P0A910 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 326 |
| Molecular Weight | 35172.3 |
| Pi | 5.59 |
| Molecular Weight | 35172.3 |
| Disulphides | Unknown |
| Full Sequence |
APKDNTWYTGAKLGWSQYHDTGFINNNGPTHENQLGAGAFGGYQVNPYVGFEMGYDWLGRMPYKGSVENGAYKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADTKSNVYGKNHDTGVSPVFAGGVEYAITPEIATRLEYQWTNNIGDAHTIGTRPDNGMLSLGVSYRFGQGEAAPVVAPAPAPAPEVQTKHFTLKSDVLFNFNKATLKPEGQAALDQLYSQLSNLDPKDGSVVVLGYTDRIGSDAYNQGLSERRAQSVVDYLISKGIPADKISARGMGESNPVTGNTCDNVKQRAALIDCLAPDRRVEIEVKGIKDVVTQPQA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Arora A, Rinehart D, Szabo G, Tamm LK. (2000) J Biol Chem, 275, 1594-600 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | MC4100 |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | n/a |
| Expression Protocol | pA and single tryptophan mutants of OmpA were expressed in the OmpA-deficient E. coli strain MC4100rh as described previously (see notes). These proteins were purified from the outer membranes by urea extraction and ion-exchange chromatography of the unfolded proteins in 8 M urea using a Q-Sepharose Fast Flow column |
| 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 | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 15 mM Tris-Cl, pH 8.5, containing 8 M urea |
| Refolding Buffer | 20 mM solution of C8E4 in 2 mM sodium borate, pH 10.0, containing 0.4 mM EDTA |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 10.0 |
| Refolding Temperature | 40.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding of OmpA into Detergent Micelles-- Refolding of OmpA and its derivatives was carried out as described in more detail by Kleinschmidt et al. (16). Briefly, 5 µl of a 4 mg/ml solution of unfolded OmpA in 15 mM Tris-Cl, pH 8.5, containing 8 M urea was diluted 50-fold into a 20 mM solution of C8E4 in 2 mM sodium borate, pH 10.0, containing 0.4 mM EDTA. The mixture was incubated overnight at 40 °C to ensure complete refolding of the protein. To remove misfolded protein aggregates the samples were centrifuged at 14,000 rpm in a table-top centrifuge (Eppendorff, Rexdale, Ontario) for 15 min prior to the addition to the planar membranes. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |