The Transcription Factor C / Ebp¦Â Functional Domains Lip Prokaryotic Expression, Purification And Functional Discussion

C/EBPs (CCAAT enhancer binding proteins) are a family of transcription factors which can bind to the enhancer region in the DNA. C/EBPβis a key member of this family with a highly conserved DNA-binding domain and dimerization domain at its C terminal. LIP is one isomer of C/EBPβ, which has only the C terminal DNA-binding domain, and has no N-terminal activation domain. In vitro, the wild-type LIP (LIPwt) has low DNA binding ability, while its mutant LIPM (Thr179/188Asp) has a strong DNA binding capacity. But the mechanism about it remains unknown. To figure out this question, we would probe the interaction between LIP and dsDNA by using NMR techniques in the future. This thesis was devided into two parts as follows, by focusing on the studies about the expression and their functions of LIPwt and its mutant LIPM.(1) The expression and purification of LIPwt and LIPM:The recombinant plasmids of SUMO-tagged pSMT3 with LIPwt and LIPM genes were constructed, and transferred into host bacteria Rossetta (DE3). The conditions for soluble expression was optimized by investigating different temperature, IPTG concentration and induction time. Then the buffer condition and chromatography methods were tried to overcome poor stability of the proteins. Finally, through nickel affinity column, CM ion exchange column and gel filtration chromatography, the NMR samples with high concentrations and stability were obtained.(2) The functional studies of LIPwt and LIPM:LIPwt was phosphorylated in vitro to get LIPwtP, three 2D 1H-15N HSQC spectra of LIPwt, LIPM and LIPwtP in free form were acquired to test the effect on the structure of LIP by (mimic) phosphorylation. Then three 2D 1H-15N HSQC spectra of LIPwt, LIPM and LIPwtP binding to dsDNA were also conducted to see the binding affinity of them to dsDNA.The results here indicated that phosphorylation has no effect on LIP 3D conformation, and all these three proteins LIPwt, LIPM and LIPwtP can binds weakly to dsDNA. But the detailed interaction models were still unclear.