PMID:9693059
| Citation |
Di Salvo, M, Yang, E, Zhao, G, Winkler, ME and Schirch, V (1998) Expression, purification, and characterization of recombinant Escherichia coli pyridoxine 5'-phosphate oxidase. Protein Expr. Purif. 13:349-56 |
|---|---|
| Abstract |
A previously cloned pdxH gene from Escherichia coli coding for pyridoxine 5'-phosphate oxidase was transferred to a pET22b vector and expressed in E. coli HMS174(DE3) cells. The soluble overexpressed enzyme was rapidly purified in high yield using two chromatography columns with an overall purification of about 2.8-fold. The purified enzyme contained tightly bound FMN. The enzyme exhibited the same spectral properties and similar kinetic constants to those previously reported by G. Zhao and M. E.Winkler (J. Bacteriol. 177, 883, 1995), but differed from the properties reported by other investigators. A rapid procedure was developed for preparing apoPNP Ox in high yield. Both the holo- and apoenzymes were homodimers. The molar absorbtivity coefficient for the protein was determined for the fully active apoPNP Ox from is amino acid composition. Using this value and the spectral properties of the bound FMN it was shown by three different methods that the dimeric enzyme contains two molecules of bound FMN per dimer and not one FMN as previously reported. |
| Links |
PubMed Online version:10.1006/prep.1998.0904 |
| Keywords |
Base Sequence; Chromatography, Gel; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Flavin Mononucleotide/metabolism; Kinetics; Protein Binding; Pyridoxaminephosphate Oxidase/genetics; Pyridoxaminephosphate Oxidase/isolation & purification; Pyridoxaminephosphate Oxidase/metabolism; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism; Structure-Activity Relationship |
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