( Introduction ...)Purification of Recombinant Flavonone 3beta-Hydroxylase Using Bio-Scale CHT5-1 Hydroxyapatite Column

Introduction
Flavanone 3b-hydroxylase (FHT), a typical 2-oxoglutaratedependent dioxygenase, catalyzes the 3b-hydroxylation of 2S-flavanones to 2R,3R-dihydroflavonols which are intermediates in the biosynthesis of flavanoids, catechins, proanthocyanidins, and anthocyanidins in plants. To understand the complex reaction mechanism, the active site of the hydroxylase, and especially its interaction with the substrates and cofactors of the enzyme reaction, knowledge of the crystal structure of the protein is required.

The E. coli-expressed enzyme from Petunia hybrida was purified to apparent homogeneity by a three-step chromatographic procedure which included rapid, high resolution chromatography on a 5 ml Bio-Scale CHT5-I ceramic hydroxyapatite column (Bio-Rad, catalog # 751-0023).

Results Recombinant flavanone 3b-hydroxylase was expressed in E. coli. The cells were lysed and the resulting supernatant from a 45% saturated ammonium sulfate precipitation step was fractionated on a butyl HIC column. The FHT-containing fraction was concentrated and further fractionated using a gel filtration column. The FHT-containing fractions from the gel filtration column were pooled and loaded directly onto a 5 ml Bio-Scale CHT5-I column. Bound proteins were eluted by a two-step linear gradient from 2550 mM in 10 ml and 50200 mM potassium phosphate in 40 ml (Figure 1). Pure enzyme was obtained in the fractions containing 5080 mM potassium phosphate. The purity of the enzyme was investigated by polyacrylamide gel electrophoresis (Figure 2).

The purification procedure was monitored by immuno dot blot analysis for FHT protein (Figure 3) and enzyme activity assays of the individual fractions after each of the chromatographic steps. The table contains the essential purification parameters.

Data courtesy of Dr Richard Lukacin, Institute for Pharmaceutical Biology, University of Marburg, Germany

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