Screening Novel HPER1 Interacting Proteins

In almost all living organisms, various physiological and behavioral functionsare expressed rhythmically with a period of about 24 hours. These daily rhythms,referred to as circadian rhythm, are controlled by self-sustained biological oscillators,which involve transcriptional-translational feedback loops of circadian genes, namely,Per1, Per2, Per3, Cry1, Cry2, Bmall, and Clock et al. In mammals, it has beenestablished that a master circadian oscillator exists in the hypothalamicsuprachiasmatic nucleus (SCN). Recently, it was also found in peripheral organs,such as liver, kidney, lung and the other tissues. As a critical component of circadian,Per1 plays an important role in resetting and sustaining circadian oscillation. But itsmechanism has not been fully clarified yet.To find novel proteins interacting with hPER1 by scanning cDNA library inhuman blood, to promote understandings about mechanisms of the circadian rhythms,yeast two-hybrid system was applied, hPER1 bHLH-PAS domain as the bait.The cDNA library of human blood was constructed according to BDMatchmaker^TM Library Construction & Screening Kits. The sequence encoding PASdomain of hPER1 was amplified by RT-PCR. The amplified fragment was ligasedwith the vector pGBKT7 to construct recombinant bait plasmid pGBKT7/hPer1_PAS.The recombinant bait plasmid is tested for transcriptional activation andtoxicity.Using sequential transformation method to construct yeast two-hybrid system(MATCHMAKER GAL4 Two-Hybrid System, BD Clontech), to screen novel hPER1interacting proteins. The transformants were selected on deficient medium and thepositive clones were tested expression of report gene throughβ-galactosidase assay.Amplified DNA fragments of the positive clones through PCR were sequenced and analyzed by bioinformatics methods.It was proved that the recombinant plasmid was constructed correctly bydigested with endonuclease and sequenced The DNA-BD Fusion is inactive andnontoxic. Forty-six positive colonies were selected by the yeast two-hybrid system.LSMD1 protein was obtained after verification.We screened a novel protein LSMD1 which can be interacted with hPER1 byusing yeast two-hybrid system.