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This version was published on June 1, 2007
Journal of Biomolecular Screening, Vol. 12, No. 4, 560-567 (2007)
DOI: 10.1177/1087057107301246

Evaluation of Different Glutathione S-Transferase–Tagged Protein Captures for Screening E6/E6AP Interaction Inhibitors Using AlphaScreen®

Peter Sehr

Peter Sehr Chemical Biology Core Facility EMBL Meyerhofstraße 1 D-69117 Heidelberg, Germany, sehr{at}embl.de

Michael Pawlita

Infection and Cancer, German Cancer Research Center, Heidelberg, Germany

Joe Lewis

Chemical Biology Core Facility, EMBL, Heidelberg, Germany

Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the development of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated protein (E6AP), based on AlphaScreen® technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the great potential of the newly developed glutathione-coated Acceptor beads as a detection reagent for GST fusion proteins. (Journal of Biomolecular Screening 2007:560-567)

Key Words: papillomavirus • E6 • GST fusion protein • AlphaScreen® • protein-protein interactions


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