Refolding Record:
| Protein | |
|---|---|
| Protein Name | Polyhydroxyalkanoate synthase |
| Abbreviated Name | PHA synthase |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Pseudomonas aeruginosa |
| UniProt Accession | O05334 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 619 |
| Molecular Weight | 67693.7 |
| Pi | 5.71041 |
| Molecular Weight | 67693.7 |
| Disulphides | Unknown |
| Full Sequence |
MATEEQSPGSGRDAQFERLNANLTRIDELSKRLTAALTKRKLSDPALHGPSSDVFLKAMT
AYMAEMMQNPAKILEHQISFWGKSLKHYVEAQHQLVKGELKPPPDVTPKDRRFSNPLWQT
HPFFNYLKQQYLMNAEAVNQAVEALEHIEPSDKKRVEYFSRQIVDLFSPTNFFGTNPDAL
ERAIATDGESLVQGLENLVRDIEANKGDLLVTLADPEAFQVGQNLATTEGSVVYRNRMFE
LIQYKPTTETVHETPLLIFPPWINKFYILDLKPQNSLLKWLVDQGFTVFVVSWVNPDKSY
AGIGMDDYIRDGYMRAMAEVRAITRQKQINAVGYCIAGTTLTLTLAHLQKAGDPSVRSAT
FFTTLTDFSDPGEVGVFLNDDFVDGIERQVAVDGILDKTFMSRTFSYLRSNDLIYQPAIK
SYMMGEAPPAFDLLYWNGDGTNLPAQMAVEYLRGLCQQDRLAGGTFPVLGSPVGLKDVTL
PVCAIACETDHIAPWKSSFNGFRQFGSTDKTFILSESGHVAGIVNPPSRNKYGHYTNEGP
LDTPAAFREGAEFHAGSWWPRWGAWLAERSGKQVPARQPGDSKHPELAPAPGSYVAAPGG
AEVAKPAAR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Rehm BH, Qi Q, Beermann BB, Hinz HJ, Steinbuchel A. (2001) Biochem J., 358, 263-268 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | unknown |
| Expression Vector | pT7-7 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 50mM phosphate, 2M urea, 0.5% Triton X-100 pH 8.0 |
| Solubilization Buffer | 50mM phosphate, 6M guanidinium chloride, 3mM betamercaptoethanol pH 8.0 |
| Refolding Buffer | 50mM phosphate, 300mM NaCl, 5mM imidazole, 1mM betamercaptoethanol, 10% glycerol. |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The washed inclusion bodies were solubilised and subjected to nickel-NTA chromatography. The matrix was equilibrated and washed with 50mM phosphate, 300mM NaCl, 5mM imidazole, 1mM betamercaptoethanol, 10% glycerol, 8M urea, pH 8.0 and the protein was refolded by application of a 8-0M urea gradient. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 27% |
| Purity | |
| Notes | |