The synthesis and initial characterization of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic stationary phase.
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.
Moaddel
Ruin
R
Department of Pharmacology, School of Medicine, Georgetown University, Washington, DC, USA. moaddelru@grc.nia.nih.gov
Price
Gerry B
GB
Juteau
Jean-Marc
JM
Leffak
Michael
M
Wainer
Irving W
IW
D002853:Chromatography, Liquid / Q000295:instrumentation*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000138:chemical synthesis* / Q000737:chemistry
D004268:DNA-Binding Proteins; C500359:DTD1 protein, human