Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer Membrane Phospholipase A |
| Abbreviated Name | OMPLA |
| SCOP Family | Outer membrane phospholipase A (OMPLA) |
| Structure Notes | |
| Organism | Neisseria meningitidis |
| UniProt Accession | Q6DN70 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 355 |
| Molecular Weight | 39819.9 |
| Pi | 8.59 |
| Molecular Weight | 39819.9 |
| Disulphides | 0 |
| Full Sequence |
FGETALQCAALTDNVTRLVCYDRIFAAQLPSSAGQEGQESKAVLNLTETVRSSLDKGEAVIVVEKGGDALPADSAGETADIYTPLSLMYDLDKNDLRGLLGVREHNPMYLMPLWYNNSPNYAPSSPTRGTTVQEKFGQQKRAETKLQVSFKSKIAEDLFKTRADLWFGYTQRSDWQIYNQGRKSAPFRNTDYKPEIFLTQPVKADLPFGGRLRMLGAGFVHQSNGQSRPESRSWNRIYAMAGMEWGKLTVIPRVWVRAFDQSGDKNDNPDIADYMGYGDVKLQYRLNDRQNVYSVLRYNPKTGYGAIEAAYTFPIKGKLKGVVRGFHGYGESLIDYNHKQNGIGIGLMFQDLDGI
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bos, M. P., Tefsen, B., Voet, P., Weynants, V., van Putten, J. P. M., Tommassen, J. (2005) Int Arch Allergy Immunol, 73, 2222-2231 |
| 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(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 2h |
| Expression Vector | pET11a |
| Expression Protocol | After induction, OMPLA expressing cells were harvested by centrifugation and underwent homogenization. Homogenate was then centrifuged(4500g, 30min). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M urea, 20mM TrisHCl, 100mM glycine, pH 8.0 |
| Refolding Buffer | 20mM ethanolamine, pH 10.8 containing 0.5 (w/v) 3-dimethyldodecylammoniopropane-sulfonate |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 10.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies, isolated from homogenate, was disolved in solubilization buffer before undergoing centrifugation(200,000g, 1h) to remove remaining membranes. The supernatant obtained then underwent a 100 fold dilution in refolding buffer. |
| Refolding Assay | Bioactivity,Sequence Analysis |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 100mM |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |