Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane porin M35 |
| Abbreviated Name | OMP M35 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Moraxella catarrhalis |
| UniProt Accession | Q3MK87 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 358 |
| Molecular Weight | 38094.9 |
| Pi | 5.21 |
| Molecular Weight | 38094.9 |
| Disulphides | Unknown |
| Full Sequence |
MKKLALATAVAALSVSAAQATPTVYGKAFLTVDANNTDTTYNSGLVQLSEDTNESGLNSNTSRIGFKGSEALNANTDVVYQLEYKIDIDADRGDNFKSRDTYLGLAHKQYGTLLAGRLTTIDDSVDFASMLEDNNVADIGPTFNAPRANNAFAYVSPEYNGTQFLAMYAFDSDTDKGGLAKDDQFGVGATYSTGPINAGATYIHYGDDSHIRLSGNYAVSPALTVGALYQISEFGVAAKNQKASPLSEGKVGDKKENTLIVSGEMKTATPWTAYGQATLIKNVAGNDGDESVGVGIGGKYAFNKATTGHVYTGYVNSERKNVKYEGSNETHKNAHKDTKFDGYKNSGFGIGAGLEYKF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Easton DM, Smith A, Gallego SG, Foxwell AR, Cripps AW, Kyd JM. (2005) J Bacteriol., 187, 6528-6535 |
| Project Aim | Identification and Characterization |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pQE30 |
| Expression Protocol | Production of recombinant M35 protein. A recombinant polyhistidine (six-His)-tagged form of the mature M35 protein (rM35) was created by cloning the amplified gene into a pQE30 plasmid (clones developed previously by Sara Gomez Gallego), expressed in Escherichia coli M15 pREP4 cells (Stratagene), and purified using a nickel-nitrilotriacetic acid immobilized-metal affinity chromatography technique. The first 20 residues of the translated amino acid sequence are predicted to be a leader peptide that is cleaved during the assembly of the mature protein, which is typical of bacterial outer membrane proteins. The cleavage site was predicted to be after the sequence AQA, and N-terminal amino acid sequencing confirmed this. The rM35 protein was therefore designed to not contain this leader peptide. E. coli clones were grown overnight at 37°C on Luria-Bertani (LB) medium containing 50 µg/ml of both ampicillin and kanamycin (Amresco). Single colonies were then grown in LB broth culture with the same concentration of antibiotics at 37°C in an orbital shaker to a relative optical density of approximately 0.7 at 600 nm. Expression of rM35 was then induced by the addition of isopropyl-ß-D-thiogalactopyranoside to a final concentration of 1 mM. Expression of the protein was allowed to occur over the next 4 h (under the same culture conditions) before harvesting of the cells by centrifugation at 4,000 x g for 20 min at 4°C. The pellets were stored at –80°C overnight before purification of the six-His-tagged protein using a nickel-nitrilotriacetic acid protein purification kit (QIAGEN), following the manufacturer\'s instructions, under denaturing conditions (8 M urea). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | 10 mM morpholinepropanesulfonic acid, 0.1 M NaCl, 0.2% (wt/vol) sodium dodecyl sulfate (SDS), pH 6.9 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 6.9 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding of recombinant M35 protein. Refolding of the recombinant M35 protein was achieved by a modification of the protocol described by Watanabe (31). Urea was removed from the protein solution by dialysis overnight against 10 mM morpholinepropanesulfonic acid, 0.1 M NaCl, 0.2% (wt/vol) sodium dodecyl sulfate (SDS), pH 6.9. This was followed by dilution with an equal volume of the same buffer including 20-mg/ml octylglucoside, to give a final concentration of 10-mg/ml octylglucoside. The protein was incubated at room temperature in this solution for at least 24 h prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis under nonreducing conditions. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |