Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system.
Here we demonstrate the use of a mammalian two-hybrid system to study protein-protein interactions. Like the yeast two-hybrid system, this is a genetic, in vivo assay based on the reconstitution of the function of a transcriptional activator. In this system, one protein of interest is expressed as a fusion to the Gal4 DNA-binding domain and another protein is expressed as a fusion to the activation domain of the VP16 protein of the herpes simplex virus. The vectors that express these fusion proteins are cotransfected with a reporter chloramphenicol acetyltransferase (CAT) vector into a mammalian cell line. The reporter plasmid contains a cat gene under the control of five consensus Gal4 binding sites. If the two fusion proteins interact, there will be a significant increase in expression of the cat reporter gene. Previously, it was reported that mouse p53 antitumor protein and simian virus 40 large T antigen interact in a yeast two-hybrid system. Using a mammalian two-hybrid system, we were able to independently confirm this interaction. The mammalian two-hybrid system can be used as a complementary approach to verify protein-protein interactions detected by a yeast two-hybrid system screening. In addition, the mammalian two-hybrid system has two main advantages: (i) Assay results can be obtained within 48 h of transfection, and (ii) protein interactions in mammalian cells may better mimic actual in vivo interactions.
Luo
Y
Y
CLONTECH Laboratories, Palo Alto, CA, USA. yluo@clontech.com
Batalao
A
A
Zhou
H
H
Zhu
L
L
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D006367:HeLa Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013539:Simian virus 40 / Q000235:genetics; D014157:Transcription Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase