Development of a Novel Test System for Screening for Antagonists of ErbB5 Receptors in Breast Cancer
Abstract
The focus of this project was to design a system for screening chemical compounds that would inhibit the dimerization of the EGFR family members ErbB2 and ErbB3 using beta-galactosidase complementation. We attempted to apply the beta-galactosidase complementation system to both ErbB2, 3 homo and heterodimerization, as well as the interaction of these receptors with downstream signaling molecules such as Grb2. We were unsuccessful in these endeavors owing largely to the unpredictability of the protein-protein interactions under investigation. A second goal of the proposed work was to design a mammalian two-hybrid system to identify proteins that interact with the cytoplasmic domain of ErbB2. Initial experiments with the beta-galactosidase system showed that the fluorescent substrate was not sensitive enough for this type of application. To circumvent these issues we designed a new complementation system based on beta-lactamase. This system is advantageous because a robust cell-permeable fluorescent substrate has recently been developed. Using the beta-lactamase system we showed that it meets several criteria which enable its use screening retroviral cDNA libraries such as: the ability to detect moderate affinity interactions (Fos-Jun), identify single retroviral integrations, and we were successful in performing a mock library experiment at dilutions up to 1:30,000.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 2003
- Accession Number
- ADA418054
Entities
People
- Helen M. Blau
Organizations
- Stanford University