A Preliminary Study On The Purification And GR Interaction Of The Circadian Clock Core Component CHRONO

CHRONO is an circadian clock element,and the structure and function of CHRONO is the main purpose of this research.This thesis mainly focuses on the representation and supplementary function of CHRONO.As a new core component of the biological clock,CHRONO not only occupies an important part in the core oscillation network,but also has similar functions with CRY,another core component of the biological clock.But the structure and function of CHRONO have not been well revealed.Since protein structures can facilitate the process of understanding the function,I started to study the preliminary characteristics of CHRONO structure based on literature reading and preliminary work in the laboratory,.The main purpose of this experiment is to express proteins in E.coli by constructing vectors,and characterize the physiological characteristics of the target proteins,in order to lay the foundation for the later structure research.In order to understand the physiological characteristics and existence status of CHRONO protein,I explored the CHRONO protein,the CHRNC protein which is the n-terminal and c-terminal fusion fragment of CHRONO,and the CHRH which is the helix region of CHRONO.At the same time,V29 H and V28 H skeleton vectors were selected to construct a series of CHRHC(111-296)proteins,which were expressed and purified after successful construction.Purified by nickel column to obtain 14 V28H-CHRHC target proteinThe purified protein was purified by molecular sieve,and the state of protein aggregation was determined by protein calibration experiment.The results showed that the condition of the V28H-CHRHC11,12 and14 protein is better,and V28H-CHRHC14 was a monomer.Finally,the secondary structure prediction and thermal stability measurement of V28H-CHRHC protein were carried out,and the results showed that the ratio of V28H-CHRHC series protein 220/208 showed an increasing trend.Compared with MBP,the extent of PCR helix gradually increased.At the same time,the content of helix was determined,and the results showed that,compared with MBP,the helix content of V28H-CHRHC1 to 11 protein increased,while the helix content of V28H-CHRHC11 to 14 protein(helix truncated region)decreased.At the same time,the experiment uses protein thermal stability to determine the stability of the target protein in solution,the results showed that the Tm value of V28H-CHRHC series proteins increasedcompared with MBP protein,indicating that the stability of the protein in solution increased,and the stability of V28H-CHRHC11,12,13,14 protein is better.Experimental data indicates that CHRONO protein may exist in a poly state.On the functional side,using IP experiment,the interaction between GR and CHRONO may have tissue specificity.At present,the research on the structure and function of CHRONO is inadequate.The research on CHRONO can help us complement and improve the core oscillation regulation network of the circadian clock.Understanding the circadian clock circuit and mechanism can help us treat some human diseases including depression,Alzheimer disease and cancer.