COMPOSITIONS, METHODS AND KITS FOR IDENTIFYING PROTEIN-PROTEIN INTERACTION DISRUPTING AGENTS,申请号EP20010271437-传众专利搜索

COMPOSITIONS, METHODS AND KITS FOR IDENTIFYING PROTEIN-PROTEIN INTERACTION DISRUPTING AGENTS

申请公布号：EP1343882(B1)

申请号：EP20010271437

申请日期：2001.12.13

申请公布日期：2004.06.23

申请人：

NATIONAL RESEARCH COUNCIL OF CANADA

发明人：ZHAO, HUI-FEN;SHEN, SHI-HSIANG

分类号：C12N15/10;(IPC1-7):C12N15/10

主分类号：C12N15/10

摘要：<p>The present invention provides compositions, methods, and kits for identifying agents that are capable of disrupting protein-protein interaction in a mammalian reverse two-hybrid system. A tetracycline repressor protein was fused with the inhibitory KRAB domain as a suppressor to tightly regulate the reverse two-hybrid system for mammalian cells. Binding of the chimeric protein to the tetracycline operator sequence provided within a promoter entirely suppressed the expression of two interactive proteins. When both an inducer and a candidate protein-protein interaction disrupting agent such as a drug were added in the cell culture together, the reporter gene was either activated by the interaction of protein-pair if the drug was ineffective or remained silent due to the disruption of the protein-protein interaction by the effective drug. The plasmids for the suppressor and the reporter were integrated into chromosomes. Constructs for the expression of interactive proteins were either stably or transiently transfected into the cells. The utility of this system for screening drugs, particularly for enzyme inhibitor drugs, was demonstrated by using two well characterized interactions of the type I receptor for TGFbeta with FKPB12 and the EGF receptor with p85. The interaction between TGFbetaBI and FKPB12 or between EGF receptor and p85 were disrupted by FK506 and kinase inhibitor AG1478, respectively. The mammalian reverse two-hybrid system of the present invention can also be used for high throughput screening of compounds that disrupt protein-protein interactions.</p>