A Study Of The In Vitro Interaction Between The Multifunctional Transcriptional Regulator CTCF And Fragment Of Vigilin

Background &Objective Research work on life science has entered the postgenome era after the accomplishment of Human Genome Project. Biologic activities of cell are mediated by proteins. Many protein-protein interactions bind the protein factors together to form functional complexes. In order to be better known to the biologic activities, it is necessary to research on the function of protein monomers and complexes which also reveals the importance of studies on protein-protein interaction. CTCF is a ubiquitously-expressed, highly-conserved, multivalent transcription factor that utilizes different zinc finger combinations to specifically bind diverse nucleotide sequences to play different roles as a regulator of expression in different genes. It is capable of both activating and repressing gene transcription, gene silencing, enhancer-blocking, imprinting control and X chromosome inactivation. CTCF regulates many aspects of cell physiological functions through its target genes, which play fundamental roles in the regulation of cell growth, proliferation, differentiation, apoptosis, heredity, epigenetics and tumor genesis. In our former study, we used CTCF as a bait to screen through a human liver cDNA pretransformed library using the yeast two-hybrid assay to search for interaction candidates of CTCF. We identified a positive cDNA clone containing fragments which partly encoding a fraction of Vigilin. In this study, we cloned and expressed the full length CTCF protein and its three fractions: N, Zn and C in E.coli. We cloned and prokaryotic expressed the Vigilin-315 and Vigilin-3KH which are both fractions of the protein Vigilin. After purifying these proteins by performing affinity chromatography assay, we used GST pull-down assay to confirm if there were interaction between CTCF/CTCF fractions and vigilin-315 fraction.Methods The recombinant plasmid pGEM7Zf (-)-CTCF encoding the full length of CTCF's cDNA sequence was gifted by Professor W.W. Quitschke. Using the plasmid as template, we constructed GST-tag fusion prokaryotic expression plasmids pGEX-4T-2-CTCF, pGEX-4T-2-CTCF-N, pGEX-4T-2-CTCF-Zn and pGEX-4T-2-CTCF-C. Then these constructs were transformed into E.coli BL21. Positive clones were acquired after identification by restriction enzyme analysis and sequencing. Fusion proteins were expressed after the host bacteria were induced with IPTG using optimized expression condition. Fusion proteins were purified with GSTrap FF affinity chromatography. Using the same strategy, we constructed His-tag fusion prokaryotic expression plasmids pET28a(+)-315, pET28a(+)-3KH. Then these two constructs were transformed into E.coli BL21(DE3). Positive clones were acquired after identification by restriction enzyme analysis and sequencing. Fusion proteins were expressed after the host bacteria were induced with IPTG using optimized expression condition. Fusion proteins were purified with HisTrap FF affinity chromatography and identified through Far-Western Blot assay. The GST fusion proteins were used as baits in the GST pull down assay to test whether there is any interaction between anyone of them and the Viglin-315 protein.Result The recombinant plasmids pGEX-4T-2-CTCF, pGEX-4T-2-CTCF-N, pGEX-4T-2-CTCF-Zn and pGEX-4T-2-CTCF-C, pET28a (+)-315 and pET28a (+)-3KH were proved correctly constructed by restriction enzyme assay and sequencing. All GST fusion proteins, CTCF, CTCF-N, CTCF-Zn and CTCF-C were successfully expressed and purified with affinity chromatography. Both His fusion proteins, Vigilin-315 and Vigilin-3KH were successfully expressed, Vigilin-315 was purified and identified via affinity chromatography and Far-Western Blot. The GST pull down assay shows that none of the GST fused CTCF and its fractions have interacted with His fused Vigilin-315 protein. Conclusion We succeeded in constructing the recombinant plasmids expressing CTCF, CTCF-N, CTCF-Zn, CTCF-C, Vigilin-315 and Vigilin-3KH. The expressions of these proteins were successful. The fusion proteins CTCF, CTCF-N, CTCF-Zn, CTCF-C and Vigilin-315 were successfully purified with affinity chromatography. The GST pull down assay shows that none of the GST fused CTCF and its fractions have interacted with His fused Vigilin-315 protein.