Refolding Record:
| Protein | |
|---|---|
| Protein Name | Pyruvate formate-lyase activating enzyme |
| Abbreviated Name | PFL |
| SCOP Family | PFL-like |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P0A9N4 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 250 |
| Molecular Weight | 28204.3 |
| Pi | 6.00179 |
| Molecular Weight | 28204.3 |
| Disulphides | Unknown |
| Full Sequence |
MSVIGRIHSFESCGTVDGPGIRFITFFQGCLMRCLYCHNRDTWDTHGGKEVTVEDLMKEV
VTYRHFMNASGGGVTASGGEAILQAEFVRDWFRACKKEGIHTCLDTNGFVRRYDPVIDEL
LEVTDLVMLDLKQMNDEIHQNLVGVSNHRTLEFAKYLANKNVKVWIRYVVVPGWSDDDDS
AHRLGEFTRDMGNVEKIELLPYHELGKHKWVAMGEEYKLDGVKPPKKETMERVKGILEQY
GHKVMF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Wong KK, Murray BW, Lewisch SA, Baxter MK, Ridky TW, Ulissi-DeMario L, Kozarich JW. (1993) Biochemistry, 32, 14102-14110 |
| 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 | N4830 |
| Expression Temp | 42.0 |
| Expression Time | 2h |
| Expression Vector | pMG27NS |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, pH 7.2 |
| Solubilization Buffer | 50mM Tris-HCl, 6M guanidinium chloride, 100mM DTT pH 7.2 |
| Refolding Buffer | 50mM Tris-HCl, 0.5mM DTT |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.2 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.1mg/ml |
| Refolding Time | 14h |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The solubilized protein was purified on Superose-12 size exclusion chromatography. Fractions containing the target protein were subsequently diluted 100-fold with 50mM Tris-HCl, 0.5mM DTT that had been purged with argon. Refolding was allowed to continue at 4 degrees C for 14h under anaerobic conditions. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 40% |
| Purity | |
| Notes | |