Purification of Recombinant Human Glucose-6-Phosphate Dehydrogenase. Lilit Nalbandyan and Ivon Brito
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The current study has been aimed at isolating and purifying appropriate amounts of the human recombinant <i style="mso-bidi-font-style: normal">Glucose-6-Phosphate Dehydrogenase from <i style="mso-bidi-font-style: normal">Escherichia coli strain HB351. Procedures have been applied for utilizing reduced volumes of concentrated Terrific Broth for growing cells at 35?C and harvesting large masses of E.coli, for the expression of the enzyme is directly related to the number of cells from which it is extracted. Sonication of the harvested cells, subjected to free/thaw technique, results in 95% cell rupturing when the process is performed with diluted cell suspensions. Affinity chromatography performed with sonicated extract allows 100% binding of the enzyme to the beads of the matrix - Affi-gel Blue Gel ? and 95-100% of the enzyme release through the treatment of the G-6PD bound beads with high concentration of a salt solution. The subsequent fractionation of the catalyst has been achieved by the precipitation of the enzyme in the affinity chromatography filtrate with ammonium sulfate. The resuspension of the precipitate pellet in small volumes of a buffer allows 75-80% catalyst recovery.The enzyme solution from ammonium sulfate precipitated step has been subjected to S-300-HR Sephacryl size-exclusion chromatography, the application of which results in the appearance of the aggregated molecule, G-6-PD, in the earlier elution fractions; the final purity of the samples from size-exclusion chromatography has been determined by SDS-PAGE, exhibiting a single band for G-6-PD. The presence of the enzyme in all of the steps has been detected by kinetic measurements.