Establishment Of Transcription Factors FOXA2 Biotin Tagging System And Identification Of Interactions Protein

Breast cancer is one of the most common cancers which threatening the female health, and the incidence of this disease has the obvious trend of ascent.Breast cancer cells have three features:unlimited growth, transformation and metastasis, making it difficult to be eliminated.Therefore,a deep probe into the signaling pathways mechanisms of breast cancer cells appreciation、migration and invasion is good for finding potential and special markers and developing therapeutical methods in the future.Biotin Avi-tag technology is a small tag that consisted of 15 amino acids (GLNDIFEAQKIEWHE) acceptor peptide, and linked to the N-teminus or C-terminus of the target protein. The tag can be specifically recognized by biotin ligase BirA that catalyzes biotin transferring to the tag’s lysine residue of the target protein, resulting in the target protein biotinylated in vivo or in vitro. The biotinylated process is an enzymatic reaction, whose conditions are quite mild and highly specific. Biotin Avi-tag technology is known as efficient, site-specific and simple. Because the tag is short, the target protein’s conformation and activity is kept same as its nature protein. Streptavidin can specifically bind with biotin, and the binding force between the streptavidin and biotin is the strongest among the known non-covalent forces in nature. Their binding force is 10,000 times higher than that of the binding between antigen and antibody. Thus, the target protein and its specifically interacting protein complexes can be isolated in a stringent elution conditions.Forkhead box (FOX) family protein was found in a lot of fungi and animals, is the main characteristic of the family as a transcription factor acts on the DNA sequence, and the tail of the family proteins has special wing helical structure, the biggest characteristic is its DNA sequence combined with regional extreme conservative in FOX family protein, FOXA2 is one of the members of the family of transcription factors. FOXA2 involved in cell cycle regulation, and inhibit the transfer of a variety of tumor cells.In numerous tumor samples such as breast, lung and found that the expression of FOXA2 has a higher level, high FOXA2 expression maybe is closely associated with cell canceration.FOXA2 also participated in a variety of signaling pathways, including regulation of carcinogenic signaling pathways.Study of protein interactions is an important research content at post-genomic period, is also the current very popular research areas.Organisms, caused by the external environment of communication signal, by ligand binding transmembrane receptors on the surface of the cell, and then through the protein-protein interaction cause certain physiological and biochemical reactions in the nucleus.Every small molecules in biological organism is not exist independently, is a overlapping network closely linked together. Cancer control can not only suppress the single signal pathway, development of tumor is made up of many different signaling pathways participation, mutual influence.By recognizing FOXA2 as the core, to find its protein interactions, analysis of signaling pathways regulate the network, it is great important for understand biology function of FOXA2.Based on biotin tagging system, Avi-tag can be biotinylated by biotin ligase (BirA) in cells. We constructed a eukaryotic expression vector FOXA2-Avi-tag. Biotin binds to streptavidin with an extremely high affinity, and high specificity. Then the protein complexes was coomassie brilliant blue staining, mass spectrometry, bioinformatics analyzed to looking for FOXA2 possible interactions proteins, and then validated by molecular biology methods, such as immune coprecipitation, in our study, we found FOXP2 is FOXA2 of interactions protein, and through the statistical analysis of clinical samples, we found FOXP2 and FOXA2 expression level with phase correlation.This study including two points:one, Established a transcription factor FOXA2 eukaryotes biotin tagging system, effective implementation Avi-FOXA2 in breast cancer cell by biotin and the protein in the Western blotting, pull-down experiments can be realized only one step without application of the antibodies, this method is rapid, economic and efficient.2. To find and verify transcription factor FOXP2 is interact with FOXA2 protein, further understanding of FOXA2 function.