title: The synthesis and initial characterization of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic stationary phase.
abstract: The DNA unwinding element binding protein (DUE-B) plays a key role in DNA replication. The DUE-B protein has been immobilized on a liquid chromatography support and the resulting immobilized protein column was used for the on-line screening of a series of steroids. The DUE-B protein was expressed with an added C-terminal sequence of six adjacent histidine residues, a His6-tag and immobilized on a chiral ligand exchange support, the CLC-L column, using Ni2+ as the coordinating metal ion. The chromatographic retentions of 12 steroids were determined on the DUE-B/CLC-L column. The magnitudes of the steroid-immobilized DUE-B interactions, reflected by the observed retention times, correlated to the effect of the steroids in the cell-free replication system, i.e. the longer the retention, the greater the increase in DNA replication. The coefficient of determination for the %DNA activities linear relation to retention time was 0.9694. The data suggest that the DUE-B/CLC-L phase can be used for on-line pharmacological studies. The results also indicated that His-tagged proteins can be directly immobilized on the CLC-L stationary phase and the resulting columns used as rapid screens for the isolation and identification of small molecule or protein ligands from complex biological or chemical mixtures.
authors.affiliation: Department of Pharmacology, School of Medicine, Georgetown University, Washington, DC, USA. moaddelru@grc.nia.nih.gov
authors.forename: Ruin
authors.initials: R
authors.lastname: Moaddel
authors.forename: Gerry B
authors.initials: GB
authors.lastname: Price
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Juteau
authors.forename: Michael
authors.initials: M
authors.lastname: Leffak
authors.forename: Irving W
authors.initials: IW
authors.lastname: Wainer
mesh_terms: D002853:Chromatography, Liquid / Q000295:instrumentation*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; C500359:DTD1 protein, human