Screening Novel HPER1 Interacting Proteins And Studying The Relationship With Circadian Rythms

Objectives:To capture novel proteins interacting with hPER1 by scanning cDNA library of human brain,to promote understandings about mechanisms and signal transduction pathways of the circadian rhythms. The whole research was divided into three parts: 1. Scanning and identification of novel hPER1 interacting proteins from cDNA library of human brain; 2. Identificated the key domains of the novel protein in protein interaction; 3. Functions'linkage between the novel protein and hPER1.Methods and results:Part 1 Scanning novel hPER1 interacting proteins from cDNA library of human brainMethods: The cDNA library of human brain was constructed according to BD Matchmaker? Library Construction & Screening Kits. The sequence encoding PAS domain of hPER1 was amplified by RT-PCR. The amplified fragment was ligased with the vector pGBKT7 to construct recombinant bait plasmid pGBKT7/hPer1PAS. Using sequential transformation method to construct yeast two hybrid system (MATCHMAKER GAL4 Two-Hybrid System,BD Clontech),to screen novel hPER1 interacting proteins. The transformants were selected on deficient medium and the positive clones were tested expression of report gene throughβ-galactosidase assay. The positive clones were also checked through Co-immunoprecipitation experiment.