The Study Of Purification And Anti-tumor Activity Of TAT-FOXM1-C(688-748) Recombinant Protein

FOXM1（Forkhead box M1）is a member of the FOX transcription factor family.It has three common subtypes,namely FOXM1a,FOXM1b,and FOXM1c.The most studied one is FOXMb.FOXM1 can mediate the transcriptional activation of downstream genes related to the cell cycle,and plays an important role in the development of embryos,cell differentiation,tissue and organ regeneration and the development of malignant tumors.More and more reports in the literature indicate that FOXM1 is one of the most common highly expressed proteins in human malignant tumors,and is closely related to tumor formation,poor prognosis,drug sensitivity and drug resistance.Abnormally high expression of FOXM1 promotes the transcriptional activation of downstream genes,which will lead to adverse consequences such as abnormal proliferation,metastasis,migration and invasion,affecting the process of tumor formation.The development and research of anti-cancer drugs with FOXM1 as the target has broad prospects and is a potential strategy for clinical treatment.FOXM1is an important biomarker for the diagnosis and treatment of cancer and is also a popular research molecule for researchers from various countries.Cell-Penetrating Peptides（CPPs）are short peptides rich in basic amino acids that can pass through the natural cell membrane barrier and enter the cytoplasm and even the nucleus without destroying the complete structure of the cell.Cell penetrating peptides have a powerful carrying function,and can be combined with various macromolecular substances（such as proteins,peptides,DNA,chemical small molecule drugs,nanoparticles,fluorescein,Adenovirus vectors,liposomes,iron particles,etc.）through chemical or genetic covalent or non-covalent binding methods,even can load proteins above 100KD without damaging the cells in the membrane.Among them,the most studied is the trans-activating transcriptional activator（TAT）derived from human immunodeficiency virus HIV.Its 47-57 amino acids play a vital role in transmembrane transport.It is a kind of Naturally derived membrane-penetrating peptides have good biocompatibility,can exhibit excellent membrane-penetrating effects in vivo,have good application prospects,and have been widely used in the development of anti-tumor drugs.In this paper,FOXM1 and its different protein segmented plasmids were co-transformed in hela cells,and a dual-luciferase reporter gene experiment was used to select a segmented FOXM1_688-748that inhibits the transcriptional activity of FOXM1.In order to determine whether it has anti-tumor activity,a p GEX-4T2-FOXM1_688-748-TAT prokaryotic expression vector was constructed using the fusion cell transmembrane peptide TAT.The prokaryotic expression system and GST tag affinity purification were used to prepare a GST tag and fuse Recombinant protein of the cell penetrating peptide TAT.Because a specific thrombin recognition site is included between the GST tag and the target protein,a recombinant protein without the GST tag containing only the target protein fusion cell transmembrane peptide TAT:TAT-FOXM1_688-748was obtained by thrombin digestion.TAT-FOXM1_688-748was directly used to treat different types of tumor cells,and it was found that it can effectively inhibit cell viability and also inhibit the transcriptional activity of FOXM1.This indicates that TAT-FOXM1_688-748has potential antitumor activity and is expected to become a drug for treating tumors.