Refolding Record:
| Protein | |
|---|---|
| Protein Name | Protein translocase SecA |
| Abbreviated Name | SecA |
| SCOP Family | Sec-C motif |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P10408 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 901 |
| Molecular Weight | 102023.0 |
| Pi | 5.43131 |
| Molecular Weight | 102023.0 |
| Disulphides | Unknown |
| Full Sequence |
MLIKLLTKVF GSRNDRTLRR MRKVVNIINA MEPEMEKLSD EELKGKTAEF RARLEKGEVL ENLIPEAFAV VREASKRVFG MRHFDVQLLG GMVLNERCIA EMRTGEGKTL TATLPAYLNA LTGKGVHVVT VNDYLAQRDA ENNRPLFEFL GLTVGINLPG MPAPAKREAY AADITYGTNN EYGFDYLRDN MAFSPEERVQ RKLHYALVDE VDSILIDEAR TPLIISGPAE DSSEMYKRVN KIIPHLIRQE KEDSETFQGE GHFSVDEKSR QVNLTERGLV LIEELLVKEG IMDEGESLYS PANIMLMHHV TAALRAHALF TRDVDYIVKD GEVIIVDEHT GRTMQGRRWS DGLHQAVEAK EGVQIQNENQ TLASITFQNY FRLYEKLAGM TGTADTEAFE
FSSIYKLDTV VVPTNRPMIR KDLPDLVYMT EAEKIQAIIE DIKERTAKGQ PVLVGTISIE KSELVSNELT KAGIKHNVLN AKFHANEAAI VAQAGYPAAV TIATNMAGRG TDIVLGGSWQ AEVAALENPT AEQIEKIKAD WQVRHDAVLE AGGLHIIGTE RHESRRIDNQ LRGRSGRQGD AGSSRFYLSM EDALMRIFAS DRVSGMMRKL GMKPGEAIEH PWVTKAIANA QRKVESRNFD IRKQLLEYDD VANDQRRAIY SQRNELLDVS DVSETINSIR EDVFKATIDA YIPPQSLEEM WDIPGLQERL KNDFDLDLPI AEWLDKEPEL HEETLRERIL AQSIEVYQRK EEVVGAEMMR HFEKGVMLQT LDSLWKEHLA AMDYLRQGIH LRGYAQKDPK QEYKRESFSM FAAMLESLKY EVISTLSKVQ VRMPEEVEEL EQQRRMEAER LAQMQQLSHQ DDDSAAAAAL AAQTGERKVG RNDPCPCGSG KKYKQCHGRL Q
|
| Notes | In this study, a series of N-terminally and C-terminally truncated variants also expressed and refolded - see paper for details |
| Expression | |
|---|---|
| Report | Matsuyama S, Kimura E, Mizushima S (1990) J Biol Chem, 265, 8760-8765 |
| 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 | None |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pMAN470 |
| Expression Protocol | no detailed provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 10mM Tris-OAc pH 7.5 |
| Solubilization Buffer | 50mM Tris-OAc pH 7.5, 6M GdnHCl |
| Refolding Buffer | 1: 50mM Tris-OAc pH 7.5, 100mM KOAc, 3mM reduced glutathione, 0.3mM oxidized glutathione, 2: 50mM Tris-OAc pH 7.5, 100mM KOAc, 1mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 1h |
| Redox Agent | GSH/GSSG/DTT |
| Redox Agent Concentration | 3mM/0.3mM/1mM |
| Refolding Protocol | Cell lysates were prepared by sonication and centrifuged (10000g, 4degC, 5min). The pellets werer washed twice with wash buffer, then solubilized with solubilization buffer. The mixure was centrifuged (350000g, 4degC, 30min) and the pellet removed. 1000pmol of the unfolded solubilized protein in 80microl of buffer was diluted with 1.2ml refolding buffer 1. After standing for 1h at 4degC, the solution was dialyzed against refolding buffer 2. |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |