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

摘要

We describe the application of the two-hybrid system to the identification of protein-protein interaction temperature-sensitive mutants. We applied this strategy to the interaction between the human CDK2 cell cycle regulator and the p21Cip1 regulatory subunit. A library of randomly generated CDK2 mutant proteins was screened for interaction with p21Cip1 at different temperatures. This approach resulted in the isolation of single point mutations in CDK2 causing temperature-sensitive interaction with p21Cip1, Our results demonstrate that the two-temperature two-hybrid screen is an efficient approach for the rational design and screening of protein-protein interaction conditional mutations.