Refolding Record:
| Protein | |
|---|---|
| Protein Name | Major outer membrane protein P.IB |
| Abbreviated Name | Porin (class3) |
| SCOP Family | Porin |
| Structure Notes | |
| Organism | Neisseria meningitidis |
| UniProt Accession | P30690 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 331 |
| Molecular Weight | 35680.5 |
| Pi | 6.76208 |
| Molecular Weight | 35680.5 |
| Disulphides | 0 |
| Full Sequence |
MASMTGGQQMGRDSSLVPSS VTLYGTIKAG VETSRSVFHQ NGQVTEVTTA TGIVDLGSKI GFKGQEDLGN GLKAIWQVEQ KASIAGTDSG WGNRQSFIGL KGGFGKLRVG RLNSVLKDTG DINPWDSKSD YLGVNKIAEP EARLISVRYD SPEFAGLSGS VQYALNDNAG RHNSESYHAG FNYKNGGFFV QYGGAYKRHH
QVQEGLNIEK YQIHRLVSGY DNDALYASVA VQQQDAKLTD ASNSHNSQTE VAATLAYRFG NVTPRVSYAH GFKGLVDDAD IGNEYDQVVV GAEYDFSKRT SALVSAGWLQ EGKGENKFVA TAGGVGLRHK F
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Qi HL, Tai JY, Blake MS (1994) Infection and Immunity, 62, 2432-2439 |
| Project Aim | Structure-Function |
| 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 | 1L of LB containing 50microg/ml ampicillin was inoculated with 10ml overnight culture and incubated at 37degC with shaking until OD600 reached 0.6-1.0. IPTG was added to the culture to a concentration of 0.3mM, which were then incubated for 30min. Rifampicin was then added to a final concentration of 0.2mg/ml and the culture was incubated a further 2h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-1.0 |
| Cell Disruption Method | Chemical |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM TrisHCl, 1mM EDTA, 100mM NaCl pH8.0 |
| Solubilization Buffer | 50mM TrisHCl, 1mM EDTA, 100mM NaCl pH8.0, 8M urea |
| Refolding Buffer | 100mM TrisHCl, 200mM NaCl, 10mM EDTA, 0.05% Zwittergen 3,14, 0.02% azide |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 10mg/ml |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were harvested by centrifugation (10000rpm, 10min) and then resuspended in 3ml per g wet weight of wash buffer. To this 8microL of 50mM PMSF was added and 80microL of 10mg/ml lysozyme. The mixture was stirred for 20min ar room temperature, with deoxycholate (4mg/g wet weight cells) being added. The mixtures were then placed in a 37degC water bath and stirred with a glass rod, 20microL of DNaseI stock solution (1mg/ml) was added per g wet weight of cells. The mixtures were then removed from the water bath and left at room temperature until the solution was no longer viscous. The mixture was then centrifuged (15000rpm, 20min, 4degC). The pellet was thoroughly washed twise with wash buffer, then resuspended in freshly prepared solubilization buffer and sonicated. The protein concentration was adjusted to less than 10mg/ml, then diluted 1:1 with 10%(w/v) Zwittergen 3,14, sonicated and loaded onto a Sepharcryl S-300 molecular sieve column previously equilibrated with refolding buffer. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |