Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cyclomaltodextrin glucanotransferase |
| Abbreviated Name | CGT |
| SCOP Family | E-set domains of sugar-utilizing enzymes |
| Structure Notes | |
| Organism | Bacillus circulans |
| UniProt Accession | P04830 |
| 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 | 725 |
| Molecular Weight | 76960.4 |
| Pi | 4.92097 |
| Molecular Weight | 76960.4 |
| Disulphides | 1 |
| Full Sequence |
MKSRYKRLTSLALSLSMALGISLPAWASPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNN
PAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVN
NTSYHGYWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAE
NGGMYDNGSLLGAYSNDTAGLFHHNGGTDFSTIEDGIYKNLYDLADINHNNNAMDAYFKS
AIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKF
ANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNHDMDRF
QVAGSGTRATEQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQ
ALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLP
AGTYSDVLNGLLNGNSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMGQPG
NIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSSGTA
SNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAK
YPSWYYDVSVPAGTKLDFKFIKKGGGTVTWEGGGNHTYTTPASGVGTVTVDWQN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hellman J, Lassila P, Mantsala P. (1995) Protein Expression and Purification, 6, 56-62 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | MC1061 |
| Expression Temp | 37.0 |
| Expression Time | unknown |
| Expression Vector | pJPH12 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris-HCl, 30mM NaCl pH 7.4 |
| Solubilization Buffer | 4.5 M urea |
| Refolding Buffer | 20mM Tris-HCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 8.0 |
| Protein Concentration | 0.13mg/ml |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The whole cell pellet was resuspended in 50mM Tris-HCl, 30mM NaCl, 1mM EDTA, 0.5% Triton X-100 pH 7.4. After centrifugation the pellet was washed three times in 50mM Tris-HCl, 30mM NaCl pH 7.4 (the first wash also contained 0.2mg/ml lysozyme). The protein was solubilized in 4.5 M urea and refolded by dialysis against 20mM tris-HCl pH 8.0. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 80% - 360mg/L culture |
| Purity | one contaminating band on SDS PAGE |
| Notes | |