Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ferric enterobactin receptor |
| Abbreviated Name | FepA |
| SCOP Family | Ligand-gated protein channel |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P05825 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 758 |
| Molecular Weight | 82106.9 |
| Pi | 5.3856 |
| Molecular Weight | 82106.9 |
| Disulphides | 1 |
| Full Sequence |
MNKKIHSLALLVNLGIYGVAQAQEPTDTPVSHDDTIVVTAAEQNLQAPGVSTITADEIRK
NPVARDVSKIIRTMPGVNLTGNSTSGQRGNNRQIDIRGMGPENTLILIDGKPVSSRNSVR
QGWRGERDTRGDTSWVPPEMIERIEVLRGPAAARYGNGAAGGVVNIITKKGSGEWHGSWD
AYFNAPEHKEEGATKRTNFSLTGPLGDEFSFRLYGNLDKTQADAWDINQGHQSARAGTYA
TTLPAGREGVINKDINGVVRWDFAPLQSLELEAGYSRQGNLYAGDTQNTNSDSYTRSKYG
DETNRLYRQNYALTWNGGWDNGVTTSNWVQYEHTRNSRIPEGLAGGTEGKFNEKATQDFV
DIDLDDVMLHSEVNLPIDFLVNQTLTLGTEWNQQRMKDLSSNTQALTGTNTGGAIDGVST
TDRSPYSKAEIFSLFAENNMELTDSTIVTPGLRFDHHSIVGNNWSPALNISQGLGDDFTL
KMGIARAYKAPSLYQTNPNYILYSKGQGCYASAGGCYLQGNDDLKAETSINKEIGLEFKR
DGWLAGVTWFRNDYRNKIEAGYVAVGQNAVGTDLYQWDNVPKAVVEGLEGSLNVPVSETV
MWTNNITYMLKSENKTTGDRLSIIPEYTLNSTLSWQAREDLSMQTTFTWYGKQQPKKYNY
KGQPAVGPETKEISPYSIVGLSATWDVTKNVSLTGGVDNLFDKRLWRAGNAQTTGDLAGA
NYIAGAGAYTYNEPGRTWYMSVNTHF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Buchanan SK (1999) Biochem Soc Trans., 27, 903-8 |
| 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 |
| Expression Temp | 37.0 |
| Expression Time | 15 hrs |
| Expression Vector | pET17b |
| Expression Protocol | Not stated |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Detergent wash followed by anion exchange chromatography in urea |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution into detergent |
| Wash Buffer | solubilization buffer plus NaCl |
| Solubilization Buffer | 7 M urea, 50 mM Tris-Cl, pH 7.5, 1 mM EDTA |
| Refolding Buffer | 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 5% Zwittergent 3-14, 1% SDS |
| Pre-Refolding Purification | Detergent wash followed by anion exchange chromatography in urea |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 5 mg/ml |
| Refolding Time | 5 min |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | A refolding strategy for FepA was developed from methods which had been used to refold various porins. Purified, unfolded FepA was diluted to a protein concentration of 10 mg/ml with TE buffer and brought to room temperature. An equal volume of 10% Zwittergent-3,14 was added to the protein solution with stirring. A 10% stock solution of sodium dodecylsulphate (SDS) was added to a final concentration of 1%, and the mixture was stirred for 5 minutes. The detergent concentrations added correspond to 500 times the critical micelle concentration (cmc) for Zwittergent-3,14 and 17 times the cmc for SDS. The protein-detergent mixture was then applied to a Sephacryl S-300 26/60 gel filtration column (Pharmacia Amersham Biotech) equilibrated with 100 mM Tris-HCl, pH 8.0 / 1 M NaCl / 0.05% Zwittergent-3,14 / 10 mM EDTA / 0.02% NaN3 (room temperature). The column was eluted at 1 ml/minute and 2 ml fractions were collected. Peak fractions were analysed for refolded protein by SDS-Page electrophoresis, as folded FepA (and other outer membrane proteins) has a faster electrophoretic mobility on polyacrylamide gels than unfolded FepA, and therefore can be distinguished from unfolded material by this technique. This occurs because the folded beta barrel is stable in SDS sample buffer and can only be completely unfolded by heating at 100ºC. Fractions containing primarily refolded protein were pooled. Folded FepA was separated from unfolded material by anion exchange chromatography (Mono-Q 10/10; Pharmacia) in a buffer containing 50 mM Tris-HCl, pH 7.5 / 1% octyl polyoxyethylene monoether (C8-POE; Bachem) / 1 mM EDTA, at 4ºC. FepA was eluted with a linear (0 – 500 mM) NaCl gradient at 1 ml/minute, and 1ml fractions were collected. Unfolded FepA eluted at 75 mM NaCl, whereas folded FepA eluted at 150 mM NaCl. Fractions containing folded FepA were pooled and concentrated to 1 mg/ml, then applied to a Sepharose S-300 26/60 column in the same buffer containing 200 mM NaCl and 0.02% NaN3 (also at 4ºC). Fractions containing pure, folded FepA were pooled. For crystallisation experiments, the detergent and buffer components were exchanged using a DEAE-Sepharose CL-6B anion exchange column. Yields range from 5-10 mg purified, refolded FepA per litre of cell culture. |
| Refolding Assay | Crystallography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 5-10 mg per lit |
| Purity | High |
| Notes | n/a |