Evaluation of Different Glutathione S-Transferase-Tagged Protein Captures for Screening E6/E6AP Interaction Inhibitors Using AlphaScreen®
Peter Sehr*,
Michael Pawlita,
Joe Lewis
European Molecular Biology Laboratory [EMBL]
* To whom correspondence should be addressed. E-mail: sehr{at}embl.de.
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Abstract |
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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 XXXX:xx-xx)
