The Expression,Purification And Interaction A Study On The Human P53 And COP1/DET1 Complex

Tumors are always threatening of human life.Finding a cure for tumors has always been the most important research content of human health science.Tumor suppressor gene p53 plays an extremely important role in the occurrence,development and regulation of tumors.In many significant cell signaling pathways,p53 can activate the expression of tumor suppressor genes and thus inhibit the occurrence of tumors,and the mutation,deletion or reduction of p53 gene often leads to the disorder of intracellular signaling pathways,resulting in cell growing out of control and becoming cancerous.Because of the high mutation frequency of p53 gene in tumors,the targeted study on p53 is an important research field for the treatment of tumors.First discovered by humans more than 30 years ago,the DET1/COP1 complexes,as one of the earliest photoprogenetic inhibitors,are the key molecules in the light signaling pathway studied deeply in plants.In mammalian cells,it has been found unexpectedly that COP1 can be used as an E3 ubiquitin ligase of p53 ubiquitination,thereby inhibiting the transcriptional activation activity of p53 and p53-dependent apoptosis.This COP1-mediated p53 degradation pathway mainly maintains the stability and reactivation of p53 after DNA damage is repaired.At the same time,COP1 can catalyze the ubiquitination and degradation of proto-oncogene transcription factor c-Jun by interacting with DET1 to form a complex.At this time,COP1 does not show the function of E3 ubiquitin ligase due to the formation of COP1 and DET1 complex,which to some extent antagonizes the carcinogenic activity of cJun.In this study,we mainly investigated the possible interaction between p53,COP1 and DET1 proteins in vitro.In this study,we used genetic engineering,protein engineering and other technical means to express human p53,COP1 and DET1 proteins in large quantities through cell culture,and obtained three target proteins by fine chromatography separation and purification,and then conducted further research on the interaction among the three proteins.First we use prokaryotes expression vector pGEX-6p-1,constructed the anthropogenic length p53 protein plasmid vector successfully.Because p53 in cell nucleus usually exist in the form of four polymers,and very easy to appear when the molecular sieve purification highly polymerize,so we tried a variety of express way in segments,over against the p53,1-104,349-398,98-398 pieces,which is expressed in E.coli receptor cells BL21,the GST column and molecular sieve chromatography purification related protein fragments.In anthropogenic COP1,DET1 full-length protein expression and purification experiment,COP1 and DET1 protein expression vector were eukaryotic cell expression vector,and build the COP1 and DET1 pcDNA3.1(+)recombinant expression plasmid successfully,using competent escherichia coli cells DH5 alpha recombinant plasmid cloning,the cloned plasmids were preliminarily screened for resistance in LB AGAR medium containing Amp,then carrying HEK293 F cell lines.COP1 and DET1 proteins were preliminarily obtained by culture,passage,collection and subsequent fine purification of transfected cells,but the expression levels were very low.On this basis,COP1,DET1 and p53 protein interactions were further studied by pull-down experiments in vitro.Through the study in this paper,we expressed and purified human p53 fragment protein successfully,and constructed HEK293 F cell line capable of stable expression of human COP1 and DET1 protein successfully,which lays a good foundation for further study on the complex and interaction between human COP1,DET1 and p53.