USE OF THE 2-HYBRID SYSTEM TO IDENTIFY PROTEIN-PROTEIN INTERACTION TEMPERATURE-SENSITIVE MUTANTS - APPLICATION TO THE CDK2 P21CIP1 INTERACTION/

We describe the application of the two-hybrid system to the identifica tion of protein-protein interaction temperature-sensitive mutants. We applied this strategy to the interaction between the human CDK2 cell c ycle regulator and the p21Cip1 regulatory subunit. A library of random ly generated CDK2 mutant proteins was screened for interaction with p2 1Cip1 at different temperatures. This approach resulted in the isolati on of single point mutations in CDK2 causing temperature-sensitive int eraction with p21Cip1, Our results demonstrate that the two-temperatur e two-hybrid screen is an efficient approach for the rational design a nd screening of protein-protein interaction conditional mutations.