Construction And Preliminary Research On Protein Interaction Of FOXM1 Based On Biotin AP-tag Technology

Biotin AP-tag technology is that a small tag consisted of 15 amino acids (GLNDIFEAQKIEWHE) acceptor peptide (AP), is 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 AP-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 Ka is 10-15 mol/L that 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.FOXM1 is a transcription factor of the Forkhead family and plays important roles in the control of cellular proliferation, senescence, DNA repair, regeneration, and tumour progression. In this study, we constructed the eukaryotic expression vector for biotin AP-tagged FOXM1 and purified the biotin-labeled FOXM1 based on the high specificity, high affinity interaction between biotin and streptavidin. This will provide a foundation for the follow-up identification of FOXM1-interacting proteins. The eukaryotic expression vectors of pcDNA-AP-FOXM1 and pcDNA-BirA were constructed respectively and both vectors were co-transfected into HEK293T cells. The expression of the biotin-labeled FOXM1 in the cells was confirmed by silver nitrate staining and Western blot with HRP-labeled streptavidin. The biotin-ylated FOXM1 was purified with streptavidin agarose beads and the efficiency of the pull-down of FOXM1-interacting proteins were also analyzed. The study found that AP-FOXM1 was biotinylated in HEK293T cells. This protein enabled an one-step method for detection and purification of FOXM1 in Western blot and Pull-down experiments without using antibodies. In addition, the eukaryotic expression vectors of pcDNA-AP-FOXM1(1-224aa) and pcDNA-AP-FOXM1(1-353aa) were constructed respectively and each of them were co-transfected with pcDNA-BirA into HEK293T cells. We confirmed that P-catenin, one of FOXM1-interacting proteins, interacted with FOXM1 at the C-terminal of FOXM1. This study provide an effective tool for studying the interaction of FOXM1 with other proteins.