Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane protein Class 5 protein |
| Abbreviated Name | Opc |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Neisseria meningitidis |
| UniProt Accession | Q51230 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | Opc-P64k chimera |
| Variants | n/a |
| Chain Length | 866 |
| Molecular Weight | 91883.9 |
| Pi | 6.27 |
| Molecular Weight | 91883.9 |
| Disulphides | Unknown |
| Full Sequence |
MKKTVFTCAM IALTGTAAAA QELQTANEFT VHTDLSSISS TRAFLKEKHK AAKHISVRAD IPFDANQGIR LEAGFGRSKK NIINLETDEN KLGKTKNVKL PTGVPENRID LYTGYTYTQT LSDSLNFRVG AGLGFESSKD SIKTTKHTLH SSRQSWLAKV HADLLSQLGN GWYINPWSEV KFDLNSRYKL NTGVTNLKKD INQKTNGWGF GLGANIGKKL GESASIEAGP FYKQRTYKES GEFSVTTRSG DVSLTIPKTS IREYGLRVGI KF MALVELKVPD IGGHENVDII AVEVNVGDTI AVDDTLITLE TDKATMDVPA EVAGVVKEVK VKVGDKISEG GLIVVVEAEG TAAAPKAEAA AAPAQEAPKA AAPAPQAAQF GGSADAEYDV VVLGGGPGGY SAAFAAADEG LKVAIVERYK TLGGVCLNVG CIPSKALLHN AAVIDEVRHL AANGIKYPEP ELDIDMLRAY KDGVVSRLTG GLAGMAKSRK VDVIQGDGQF LDPHHLEVSL TAGDAYEQAA
PTGEKKIVAF KNCIIAAGSR VTKLPFIPED PRIIDSSGAL ALKEVPGKLL IIGGGIIGLE MGTVYSTLGS RLDVVEMMDG LMQGADRDLV KVWQKQNEYR FDNIMVNTKT VAVEPKEDGV YVTFEGANAP KEPQRYDAVL VAAGRAPNGK LISAEKAGVA VTDRGFIEVD KQMRTNVPHI YAIGDIVGQP MLAHKAVHEG HVAAENCAGH KAYFDARVIP GVAYTSPEVA WVGETELSAK ASGRKITKAN FPWAASGRAI ANGCDKPFTK LIFDAETGRI IGGGIVGPNG GDMIGEVCLA IEMGCDAADI GKTIHPHPTL GESIGMAAEV ALGTCTDLPP QKKK
|
| Notes | Opc-P64k protein fusion. P64k, also from N.meningitidis, acts as a stabilizer, |
| Expression | |
|---|---|
| Report | Musacchio A, Carmente T, Delgado M, Gonzalez S (1996) Vaccine, 15, 751-758 |
| Project Aim | Drug Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | W3110 |
| Expression Temp | 37.0 |
| Expression Time | 12h |
| Expression Vector | not stated |
| Expression Protocol | Cells were grown for 12h in 1.5L of M9 medium supplemented with 1% casein hydrolysate and 1% glucose |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | HPLC |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 1% Triton X-100, 10mM Tris, 6mM EDTA, 4M urea |
| Solubilization Buffer | 6M GdnHCl, 20mM citrate-phosphate pH 2.6 |
| Refolding Buffer | 1: 1M urea, 400microg/ml PEG, 0.2M L-arginine 2: 0.5M GdnHCl, 400microg/ml PEG, 0.4M L-arginine |
| Pre-Refolding Purification | HPLC |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.1mg/ml |
| Refolding Time | 120h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were disrupted by French press at 1g/ml in TE buffer (10mM Tris, 6mM EDTA), insolubule material was collected by centrifugation (18000g) and washed with wash buffer using a homogenizer. The protein was then dissolved in solubilization buffer and purified by HPLC using a reverse phase C4 column. The protein was freeze-dried and stored at 4degC. The pur protein was resuspended at 1mg/ml in 5M urea, then diluted in a range of various buffers containing 0.5M GdnHCl or 1M urea, along with various combinations of different compounds. The most successful buffers for renaturation are listed under refolding buffer. |
| Refolding Assay | Western Blot,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | .2/.4M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |