Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane protein OprN |
| Abbreviated Name | oprN |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Pseudomonas aeruginosa |
| UniProt Accession | A6V4W8 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 473 |
| Molecular Weight | 51320.4 |
| Pi | 5.25 |
| Molecular Weight | 51320.4 |
| Disulphides | Unknown |
| Full Sequence |
MIHAQSIRSGLAPTLGLFSLLALSACTVGPDYRTPDTAAAKIDATASEPYDRSRFESLWWKQFDDPTLNQLVEQSLTGNRDLRVAFARLRAARALRDDVANDRFPVVTSRASADIGKGQQPGVTEDRVNSERYDLGLDTAWELDLFGRIRRQLESSDALSEAAEADLQQLQVSLIAELVDAYGQLRGAQLREKIALSNLENQKESRQLTEQLRDAGVGAELDVLRADARLAATAASVPQLQAQAERSRHRIATLLGQRPEELTVDLSPRDLPAISKALPIGDPGELLRRRPDIRAAERRL
ASSTAEVGVATADLFPRVSLSGFLGFTAGRGSQIGSNAARAWSVGPSISWAAFDLGSVRARLRGARADADAALASYEQQVLLALEESANAFSDYGKRQERLVSLVRQSEASRAAAQQAAIRYREGTTDFLVLLDAEREQLSAEDAQAQAEVELYRGIVAIYRSLGGGWQPSA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lambert O, Benabdelhak H, Chami M, Jouan L, Nouaille E, Ducruix A, Brisson A. (2005) J Struct Biol., 150, 50-57 |
| Project Aim | Vaccine studies |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | C34(DE3) |
| Expression Temp | 30.0 |
| Expression Time | 2 h |
| Expression Vector | pBAD33 |
| Expression Protocol | Proteins were expressed in E. coli strain C43 (DE3) (Miroux and Walker, 1996). Single colonies were picked up and cultures were grown overnight at 37 °C in 500 ml LB medium containing 25 μg/ml chloramphenicol. The overnight cultures were sub-cultured in 5 L LB medium containing chloramphenicol and allowed to grow at 30 °C until they reached an OD600 = 2. Cells were induced by the addition of arabinose (final concentration 2 mM), grown for 2 h, and harvested by centrifugation at 8000g for 30 min. The cell pellet was resuspended in 45 ml buffer containing 20 mM Tris–HCl, pH 8, 5 mM MgCl2 and 50 U benzonase (Promega). Cells were broken by a French pressure cell at 10 000 psi and centrifuged twice for 30 min at 8500g to remove the inclusions bodies and unbroken cells. |
| Method of Induction | Arabinose |
| Cell Density at Induction | OD 2.0 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 mM Tris-HCl, pH 8, 10% glycerol, 2% βOG (Anatrace) |
| Refolding Buffer | 20 mM Tris–HCl buffer, pH 8 without salts, containing 0.9% βOG (Octyl-βd-glucopyranoside) |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet corresponding to the outer membrane fraction was resuspended in a solution containing 20 mM Tris-HCl, pH 8, 10% glycerol, 2% βOG (Anatrace), and stirred overnight at 23 °C. The solubilized membrane proteins were recovered by centrifugation for 30 min at 50 000g and loaded onto a Ni–NTA resin column pre-equilibrated in 20 mM Tris, pH 8, 10% glycerol, 0.9% βOG (buffer A). The column was washed with buffer A plus 10 mM imidazole. The protein was eluted with a linear gradient of imidazole (10–400 mM) at a flow rate of 5 ml/min. The fractions containing the OprM or OprN proteins eluted between 100 to 250 mM imidazole. They were pooled, concentrated and exchanged for a suitable buffer by gel filtration chromatography. Finally, each protein was concentrated at 5 mg/ml on “Amicon Ultra” cut-off 30 000 (Millipore) in the presence of 10% glycerol, 20 mM Tris-HCl, pH 8.0, 0.9% βOG, and 0.03 mM imidazole. Protein concentration was determined using the Coomassie Plus Protein Assay Reagent “OD595 nm” (PIERCE) method. Samples were analyzed on acrylamide gels (Laemmli, 1970), using a 14% (w/v) acrylamide resolving gel and a 4% stacking gel. Gels were stained using Coomassie brillant blue. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 2 mg/L |
| Purity | n/a |
| Notes | n/a |