The Study Of Scale Purification And Anti-Tumor Efficacy Of R9-FOXM1-N Recombinant Protein

FOXM1 gene,widely distributed in various eukaryotic organisms,are involved in the regulation of cell differentiation,proliferation,metabolism and apoptosis and other physiological process.FOXM1 was first recognized as a key regulator in both cell cycle and cell proliferation.FOXM1 has been demonstrated to regulate the transcription of multiple genes related to cell cycle.It is also involved in the cell proliferation,DNA replication,mitotic process,and the repair of DNA damage.Moreover,FOXM1 is established as a novel factor involved in the maintenance of cell stem.The inhibition of FOXM1 results in the absence of inducible pluripotent stem cells(iPSCs),indicating that FOXM1 is a required factor in iPSCs reprogramming.Furthermore FOXM1 has been found to be the key molecule to stimulate the epithelial mesenchymal transition of tumor cells.The inhibition of FOXM1 can prevent metastasis of cancer cells.Knockout of FOXM1 in can inhibit the occurrence and development of solid tumors such as liver cancer,lung cancer and colorectal cancer.Recent studies suggest that FOXM1 is a potential target for drug development in cancer treatment.Cell penetrating peptides are peptides composed of amino acids with the ability to penetrate the cell membrane,which earlily found from HIV-1 TAT protein contained with special peptide region.Otherwise,other natural proteins have also been found to contain peptide transmembrane region.On this basis,researchers have been screened and developed chimeric transmembrane and synthetic peptide(such as poly arginine peptides,R8 or R9),which can be modified to improve the stability and efficiency.Because of its transmembrane ability,cell penetrating peptides are considered to be mediated for biologically active substances,especially proteins and nucleic acid molecules with large molecular weight.This delivery method has high transfection efficiency and cell type of object wide,low cytotoxicity and other prominent advantages.Therefore,anti-tumor drugs can effectively act on specific targets through CPPs delivery.In this paper,we constructed a plasmid expressed the FOXM1-N terminal(1-234aa)with the CMV promoter in tumor cells,which could effectively inhibit the transcriptional activation of FOXM1.A recombinant protein expression vector was constructed and transformed to E.coli to generate a prokaryotic expression system.The recombinant protein R9-FOXM1(1-234aa)(R9-FOXM1-N)was isolated at a large scale through His-tag affinity chromatography.In vitro EMSA assay,we confirmed that R9-FOXM1-N protein could inhibit the binding of FOXM1 to its DNA binding site,suggesting that R9-FOXM1-N has the potential to inhibit FOXM1 transcriptional activity.Further processing by direct tumor cell R9-FOXM1-N,cell lysate was prepared by Western Blotting detection of the recombinant protein,proved that R9-FOXM1-N can effectively enter the cell and inhibit transcriptional activity of FOXM1 in tumor cells.We treat different types of tumor cells with R9-FOXM1-N,The results demonstrated that R9-FOXM1-N suppressed the proliferation of cancer cells,might be considered as a potential reagent for cancer treatment in the future.