Expression,Purification,And Protein-Interaction Analysis Of Human FOXA1 Protein

The Forkhead box (FOX) transcription factor family is widely distributed in various eukaryotes from yeast to human and constitutes more than 40 members in mammals, and is involved in regulating cell differentiation, proliferation, metabolism, apoptosis, and other physiological processes. The DNA binding domain (DBD) of FOX proteins family is evolutionarily conserved and contains about 100 amino acids. According to the homology of DBD amino acid sequence, the FOX proteins can be classified to 19 sub family. At present, the study of the functions and molecular mechanisms of FOX proteins has gradually become the hotpot in immunology, medical genetics, and oncology.FOXA1 transcription factor is the first cloned member of FOX family and belongs to the FOXA subfamily. FOXA1 protein contains 472 amino acids. Its functional regions can be divided into transcriptional activation domain and DNA binding domain. Its DNA domain region allowes FOXA1 to bind to promoters, and activates or represses the transcription of its target gene. FOXA1 participates in regulating the cell signal transduction, cell cycle regulation, and metabolic regulation, and it plays an important role in development and physiological processes. The functions of FOXA1 can also be affected by its interaction with other protein.To identify proteins interacting with transcription factor FOXA, we constructed different prokaryotic expression vectors for human FOXA1 protein and the proteins of FOXA1 C-terminus and FOXA1 N-terminus were purified. They provided a material foundation for future studies of FOXA1-interacted proteins. In our research, total RNAs were extracted from human breast cancer MCF7 cells and reverse transcribed into cDNAs. The full length cDNA of FOXA1 was amplified by PCR, and the C-terminal and N terminal segments were also amplified respectively. The different FOXAl cDNA fragments were cloned into prokaryotic expression vector pGEX-4T-1. The correct vectors were confirmed by double restriction enzyme digestion and DNA sequencing, and transformed into E. coli BL21 Rosetta DE3. The FOXA1 proteins were purified with Glutathione Sepharose 4B and analyzed by SDS-PAGE electrophoresis and Western Blot. GST Pull-down experiments were performed with lysates of MCF7 cells and the interaction between FOXA1 and a known FOXA1-interacted protein TLE3 was confirmed. The results indicated that the puried recombinant protein could be used as an effective material for searching the potential FOXA1 interacted proteins.