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| Refolding Record [Created: 2008-12-10 Updated: 2008-12-10 ] |
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PROTEIN |
| Protein Name |
Outer membrane protein A |
| Abbreviated Name |
OmpA |
| SCOP Family |
Outer membrane protein |
| Structure Notes |
Structure of transmembrane domain (residues 1-171) only solved |
| Organism |
Escherichia coli |
| UniProt Accession |
P0A910 |
| SCOP Unique ID |
56928 |
| Structure Solved |
y |
| Class |
Membrane and cell surface proteins and peptides |
| Molecularity |
Unknown |
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CONSTRUCT |
| Full Length |
y |
| Domain |
n/a |
| Chimera |
n/a |
| Variants |
n/a |
| Chain Length |
326 |
| Molecular Weight |
35172.3 |
| pI |
5.59 |
| Disulphides |
Unknown |
Pfam Domain(s) and
Number of Occurrences |
Pfam Entry
OmpA(1), OmpA_membrane(1) |
| Domain Graphics |
 |
| Full Sequence |
APKDNTWYTGAKLGWSQYHDTGFINNNGPTHENQLGAGAFGGYQVNPYVGFEMGYDWLGRMPYKGSVENGAYKAQGVQLTAKLGYPITDDLDIYTRLGGM
VWRADTKSNVYGKNHDTGVSPVFAGGVEYAITPEIATRLEYQWTNNIGDAHTIGTRPDNGMLSLGVSYRFGQGEAAPVVAPAPAPAPEVQTKHFTLKSDV
LFNFNKATLKPEGQAALDQLYSQLSNLDPKDGSVVVLGYTDRIGSDAYNQGLSERRAQSVVDYLISKGIPADKISARGMGESNPVTGNTCDNVKQRAALI
DCLAPDRRVEIEVKGIKDVVTQPQA
BLAST against Refold database | BLAST against NCBI NR database
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| Notes |
n/a |
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EXPRESSION |
| Report |
Arora et al., ( 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 Temperature |
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) |
n/a |
| Cell Disruption Method |
Not stated |
| Lytic Agent |
None |
| Pre-refolding Purification |
Ion-exchange chromatography |
| Solubility |
insoluble |
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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 |
| Refolding Temperature |
40 |
| 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. |
| Assay |
SDS-PAGE |
| Chaperones |
None |
| Additives |
None |
| Additives Concentration |
n/a |
| Refolding Yield |
n/a |
| Purity |
n/a |
| Notes |
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