Western Illinois University, Department of Chemistry, Macomb, IL 61455
The mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine. Hypusine synthesis in eIF5A is a posttranslational modification catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). This modification is essential for the function of eIF5A in cell proliferation and survival. It has been suggested that posttranslational modifications of eIF5A could be a suitable target for anticancer therapy. A detailed knowledge of structure-function relationship of either enzyme will help in designing better inhibitors thus better control of proliferative diseases.
Recently, we have cloned and expressed functional bovine DOHH (bDOHH) into an expression vector, pGEX-2T. (Huang et al., Protein Expr. Purif. 54, 126-133, 2007). Here we report large scale purification of bDOHH. The GST-tagged DOHH fusion protein was overexpressed by IPTG induction from 8 liters of culture and the fusion protein purified by affinity chromatography with a glutathione agarose column. The GST tag was removed by digestion with thrombin and the progress of the digestion was checked by SDS-PAGE. The bDOHH protein was then separated from the GST carrier by glutathione agarose affinity chromatography following dialysis and ultrafiltration. The monomeric bDOHH protein was then separated from aggregates by gel filtration using a Superdex 200 column. The purified protein is being used for initial crystallization screening.
[Abstract (DOC)]