EC Number   |
|---|
    1.5.1.30 | - |
| 1.5.1.30 | bifunctional enzyme flavin reductase (NADPH)/biliverdin-IXbeta reductase |
| 1.5.1.30 | cells harvested by centrifugation, washing twice and suspended in 50 mM Tris-HCl buffer, pH 8.0, with 1 mM dithiothreitol and 10% glycerol, cells disrupted with ultraoscillator (20 kHz), debris removed by centrifugation, enzyme purified from cell-free extracts with ÄKTA explorer, applied to Toyopearl DEAE-650 M column, equilibrated with 50 mM Tris-HCl buffer, pH 8.0, washed, proteins eluted with linear gradient of 0-0.5 M KCl, active fractions (at 0.15-0.2 M KCl) combined and concentrated by ultrafiltration (membrane filter YM-30), enzyme purified with HiLoad 16/60 Superdex 200, equilibrated with 50 mM Tris-HCl buffer, pH 8.0 with 0.15 M NaCl, elution with same buffer |
| 1.5.1.30 | crude lysate assay with briefly centrifuged cells, resuspended in B-PER bacterial protein extraction reagent, recombinant pZCFRE1 expression product is clarified by centrifugation and applied to a custom nickel affinity column, dialyzed into buffer containing 100 mM Na+/K+ phosphate and 100 mM NaCl, pH 7.0 |
| 1.5.1.30 | FMN-agarose chromatography as a key step |
| 1.5.1.30 | HiTrap DEAE column chromatography, Mono Q column chromatography, and Superdex 75 gel filtration |
| 1.5.1.30 | preparation of apoenzyme |
| 1.5.1.30 | recombinant enzyme 23fold from Escherichia coli to homogeneity by dialysis, anion exchange chromatography, dialysis, two steps of Blue Sepharose affinity chromatography, and again dialysis |
| 1.5.1.30 | recombinant enzyme from Escherichia coli strain BL21(DE3) to over 90% purity, recombinant fusion protein from Escherichia coli strain JM109 to over 80% purity |
| 1.5.1.30 | recombinant enzyme from Escherichia coli to homogeneity |
