The orphan receptor SHP and the three-hybrid interference assay.
The three-hybrid interference method described here is similar to previous interference assays based on the two-hybrid system. In these approaches the presence of a third protein disrupts the interaction between the two different hybrid proteins. The three-hybrid interference assay circumvents a potential problem that arises when the third protein is itself a direct transcriptional repressor by preventing the decrease in transcriptional readout that could occur if the third protein is recruited indirectly to the two-hybrid protein complex. This three-hybrid method should be applicable to any third protein that is a transcriptional repressor. To use this method to explore the effects of such repressors on protein-protein interactions, however, it is important to confirm that the VP16-repressor fusion does not retain active repressor function. It is also appropriate to compare the effects of the VP16-repressor fusion to those with the repressor alone. Finally, it is essential to confirm any conclusions from this or any other single method to study protein-protein interactions with alternative independent approaches.
Lee
Yoon-Kwang
YK
Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
Moore
David D
DD
D000818:Animals; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D019384:Nucleic Acid Synthesis Inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; C099713:nuclear receptor subfamily 0, group B, member 2; D005047:Etoposide