Refolding Record:
| Protein | |
|---|---|
| Protein Name | PorB (class 3) porin |
| Abbreviated Name | PorB |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Neisseria meningitidis |
| UniProt Accession | P30688 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 336 |
| Molecular Weight | 35740.7 |
| Pi | 6.45831 |
| Molecular Weight | 35740.7 |
| Disulphides | Unknown |
| Full Sequence |
MKKSLIALTLAALPVAAMADVTLYGTIKAGVETSRSVEHNGGQVVSVETGTGIVDLGSKI
GFKGQEDLGNGLKAIWQVEQKASIAGTDSGWGNRQSFIGLKGGFGKLRVGRLNSVLKDTG
DINPWDSKSDYLGVNKIAEPEARLISVRYDSPEFAGLSGSVQYALNDNAGKYNSESYHAG
FNYKNGGFFVQYGGAYKRHVRVDENVNIEKYQIHRLVSGYDNDALHASVAVQQQDAKLVE
DNYSHNSQTEVAATLAYRFGNVTPRVSYAHGFKGSFDDADLSNDYDQVVVGAEYDFSKRT
SALVSAGWLQEGKGENKFVSTAGGVGLRHKF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Song J, Minetti CA, Blake MS, Colombini M. (1998) Biochim Biophys Acta., 1370, 289-298 |
| Project Aim | Functional Studies |
| 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 | 2.5h |
| Expression Vector | pET17b |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 0.5% deoxycholate, pH 8.0 |
| Solubilization Buffer | 8M urea |
| Refolding Buffer | 100 mM Tris-HCl, 200 mM NaCl, 10 mM EDTA, 20 mM CaCl2, and 0.05% Zwittergent 3-14 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 4 mg/ml or less |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Equal volumes of urea-dissolved IBs and 10% Zwittergent 3-14 (Calbiochem) were combined and the final porin extract applied to a Sephacryl S-300 (5 ?100 cm) column (Pharmacia Biotech Inc.) equilibrated in a buffer comprised of 100 mM Tris-HCl, 200 mM NaCl, 10 mM EDTA, 20 mM CaCl2, and 0.05% Z 3-14, pH 8.0. Fractions containing class 3 protein were identified by SDS-PAGE, pooled, and applied to a Hiload Q-Sepharose HP ion exchange (2.6 ?20 cm) column (Pharmacia) equilibrated in 25 mM Tris-HCl, 200 mM NaCl, 1.0 mM EDTA, and 0.05% Z 3-14 pH 8.0. A gradient of 0.2-1.0 M NaCl was applied and class 3 protein eluted as a single peak. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 50mg/L culture |
| Purity | few feint bands on SDS PAGE |
| Notes | |