Refolding Record:
| Protein | |
|---|---|
| Protein Name | Erythromycin C-12 Hydroxylase |
| Abbreviated Name | EryK |
| SCOP Family | Cytochrome P450 |
| Structure Notes | |
| Organism | Saccharopolyspora erythraea |
| UniProt Accession | P48635 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 403 |
| Molecular Weight | 43759.4 |
| Pi | 4.8336 |
| Molecular Weight | 43759.4 |
| Disulphides | 0 |
| Full Sequence |
MTTIDEVPGMADETALLDWLGTMREKQPVWQDRYGVWHVFRHADVQTVLRDTATFSSDPT
RVIEGASPTPGMIHEIDPPEHRALRKVVSSAFTPRTISDLEPRIRDVTRSLLADAGESFD
LVDVLAFPLPVTIVAELLGLPPMDHEQFGDWSGALVDIQMDDPTDPALAERIADVLNPLT
AYLKARCAERRADPGDDLISRLVLAEVDGRALDDEEAANFSTALLLAGHITTTVLLGNIV
RTLDEHPAHWDAAAEDPGRIPAIVEEVLRYRPPFPQMQRTTTKATEVAGVPIPADVMVNT
WVLSANRDSDAHDDPDRFDPSRKSGGAAQFSFGHGVHFCLGAPLARLENRVALEEIIARF
GRLTVDRDDERLRHFEQIVLGTRHLPVLAGSSPRQSA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lambalot RH, Cane DE, Aparicio JJ, Katz L (1995) Biochemistry, 34, 1858-1866 |
| 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 | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 5h |
| Expression Vector | pLM1 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | unknown |
| Solubilization Buffer | 8M urea, 50mM Tris-HCl, 10mM DTT, pH 7.5 |
| Refolding Buffer | 50mM Tris-HCl |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 2.5mg/ml |
| Refolding Time | 16h |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The insoluble pellet (4 g) was then solubilized in 30 mL of 8 M urea, 50 mM Tris-HCl, and 10 mM DTT, pH 7.5 (solubilization buffer). Centrifugation for 15 min at 15000g removed 1 g of urea-insoluble material. The solubilized material was loaded onto a 30-mL QSepharose column which had been equilibrated with solubilization buffer. The column was washed with 200 mL of solubilization buffer (0.35 ` i n ) followed by elution with a 300-mL linear gradient of 0-250 mM NaCl in solubilization buffer. Apo-EryK began to elute at 50 mM NaCl and continued for 10 7-mL fractions. These fractions were pooled and diluted 4-fold with 50 mM TrisHCl, pH 7.5, to yield a 2.5 mg/mL protein solution (Bradford assay). Refolding of apo-EryK was achieved by dividing this 350- mL solution equally among five 4 x 45 cm dialysis membranes (Spectropor; MW cutoff, 6000-8000; 5 mL/cm capacity) and dialyzing overnight at 4 `C against 3.5 L of 50 mM Tris-HC1, pH 7.5, sparged continuously with argon. The buffer was changed once and dialyzed for another 4 h under the same conditions. The renatured protein was then dialyzed against 3.5 L of 0.1 M histidine and 20% (w/v) glycerol, pH 8.0 (reconstitution buffer), for 6 h at 4 `C with continuous argon sparging. In this manner approximately 200 mg of renatured apo-EryK was obtained. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 200mg/L culture |
| Purity | |
| Notes | |