Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cyclodextrin glucanotransferase |
| Abbreviated Name | CGTase |
| SCOP Family | alpha-Amylases, C-terminal beta-sheet domain |
| Structure Notes | |
| Organism | Thermococcus sp.B1001 |
| UniProt Accession | Q9UWN2 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 739 |
| Molecular Weight | 83244.9 |
| Pi | 4.78 |
| Molecular Weight | 83244.9 |
| Disulphides | 0 |
| Full Sequence |
MRSRKIYMLV VLLLFLGFSE QISTVAAGVS PSYPAGDPQT WVIYQIVIDR FYDGNTSNND PLKSPGLYDP NKENWKLYWG GDLEGIIAKL PYLYELGVSA IWISPVFDNI DVPINGSNGL EAGYHGYWPK DFKVIEEHFG TWEIFRRLSQ EAAKYNITII IDFVPNHSNP NDAGEYGALY DNGTFVIDYP TDANYATVHP
ITKSLSYIYN HNGGITNWND RWEVRYKNLF NLADLNQLNP WVDNYLKEST VSYLEAGIGG IRIDAVKHLE PGWLKTYADY VYARKNVFMF GEWYQGFNDE MYWDMVKFAN YTGIGLINIP LQQVLVDVFA YDTKTMWDLE SAVNKYTIDF MWQNKLTIFV DSHDVPRFLS LRNDLIRFHQ ALAFVLTAPG IPIIYYGDEQ
YLHNDTVNDF GQVGGDPYNR PMMKTWNTST TAFKLIKTLA SLRRYNPALA YGLIATRYVS DDIYIFERKF FDNVVLVAIN RNLNSPYVVS NVYTSLPDGS YNDYLGGLLN GVGITVSSGT FSVELPAGSV SVWQYKATPT DPWVGAIDPV MGRAGNIVTV SGEGFGDVPG RVLITNGQDY WTAEVTYWSD KSVEFIVPSG
ITTQLNENHV EVRIERADGA TSNGIAFEYL TNKQIPAIFE VRNTQGTNLE TQVGEFLWLT GSVPELSYWS PETIKAVGPM LCPGWPDWFV VASVPADTYI EFKFLKAPLG GTGIWEVGSN HAYLTPSSGI GEVSVEANR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Yamamoto T, Shiraki K, Fujiwara S, Takagi M, Fukui K, Imanaka T (1999) Biochem Biophys Res Commun., 265, 57-61 |
| 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(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET21a |
| Expression Protocol | Cells were grown at 37degC, when OD660 reached 0.45, gene expression was induced with 1mM IPTG and cells were grown for a further 4h before being harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 660 = 0.45 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM TrisHCl, 30mM NaCl pH 8.0 |
| Solubilization Buffer | 6M urea, 50mM TrisHCl , 1mM DTT pH 8.0 |
| Refolding Buffer | 50mM TrisHCl, 1mM DTT, pH 8.0 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Cells were sonicated in 50mM TrisHCl pH 8.0, and the insoluble fraction was recovered by centrifugation. The insoluble fraction was treated with 2% Triton X-100, 10mM EDTA (pH 8.0)and sonicated again. Inclusion bodies were then collected by centrifugation and washed twise with wash buffer. The suspension was then resuspended in solubilization buffer for 1h. The protein was refolded by dialysis against refolding buffer, then purified by anion-exchange chromatography (Q sepharose) and gel filtration. |
| Refolding Assay | SDS-PAGE,Native PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |