Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane protein 21 |
| Abbreviated Name | Omp21 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Comamonas acidovorans |
| UniProt Accession | O86137 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | Residues 9-183 of mature Omp21 |
| Chain Length | 187 |
| Molecular Weight | 20589.3 |
| Pi | 9.05 |
| Molecular Weight | 20589.3 |
| Disulphides | 0 |
| Full Sequence |
VRVRAVNLDSANKDSTGLGLSVINNKWMPELDVSYFFTPNIAAELVLTYPQKHDLRANGLGQIGSLKHLPPTLLAQYHFTNFGAFKPYVGAGVNYTRFSSVSFNPAVQAALNPSIKKNSFGGALQIGFDYALDKNWSLNFDVKKVFIETDVSSAGTKVGTFKVNPVLVGVGLGYRFDDDDKHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Baldermann, C., Engelhardt, H. (2005) J Struct Biol, 131, 96-107 |
| Project Aim | Crystallography,Recombinant Protein Expression,Protein refolding |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 |
| Expression Vector | pET-22b |
| Expression Protocol | Induced cells, grown for four hours, were harvested via centrifugation(3500g, 15min, 4degC). Cells r |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 1 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 8M urea, 0.1 M NaH2PO4, 0.01 M Tris, pH 8.0 |
| Solubilization Buffer | Laemlli solubilisation buffer |
| Refolding Buffer | 100mM Hepes, 200mM NaCl, 3mM NaN3, pH 7.5, 1% CeEn |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 990min |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolded while bound to a NiNTA column, equilibrated with wash buffer at pH 8.0 then with wash buffer at pH 6.3. Wash buffer was then exchanged for refolding buffer (without 1% C8En). Refolding occured via slowly exchanging refolding buffer without C8En for refolding buffer with C8En. Refolded protein was then eluted with imidazole in refolding buffer and dialysed against 1mM TrisHCl, pH 8.0, 3 mM NaN3 and 0.5% C8En. |
| Refolding Assay | Crystallography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 7 mg/ml |
| Purity | n/a |
| Notes | n/a |