Expressed, Purified, Identified And Biological Fuction Analysised Of Human Foxp3 Fusion Proteins

FOXP3,a member of the forkhead-winged-helix family of transcriptional regulators,which has been touted as a lineage-specific marker of regulatory T cells(Tregs).It has been shown to be a key molecule necessary and sufficient for Tregs development and function. FOXP3 mutations might result in a defect in dominant tolerance mediated by regulatory T cells.Recently some research work had demonstrated that ectopic expression of FOXP3 could convert effector T cells into Treg-like cells phenotypically and functionally.The protein transduction domain(PTD),a part of transactivator(TAT) protein encoded by the human immunodeficiency virus type 1,is capable of mediating heterologous protein transduce into the cytoplasm or nucleus of almost all eukaryotic cells freely and effectively,which doesn't affect the function of fusion proteins.Objective:To express and purify PTD human FOXP3 (PTD-hFOXP3),PTD-eGFP-human FOXP3(PTD-eGFP-hFOXP3) fusion proteins and to detect their transduction effiency and their effect on T cells proliferation.Methods:DNA sequences of hFOXP3 was inserted into pET28a -PTD and pET28a-PTD-eGFP vector respectively.The PTD-hFOXP3 and PTD-eGFP-hFOXP3 fusion proteins were expressed in E.coli Rosetta (DE3) and purified via Bio-Rad Profinity IMAC Ni-Charged Resin,at the end identified by Western-blot.The transmembrane effiency of fusion proteins were analysed by using FACS,western-blot and confocal.The effect of fusion proteins on the proliferation of T cell was assayed by Cell Counting Kit-8(CCK-8).Results:Prokaryotic expression vectors of pET28a-PTD-FOXP3, pET28a-PTD-eGFP-hFOXP3 were constructed correctly.The fusion proteins were expressed and purified efficiently.The results of FACS, confocal and western-blot indicated that fusion proteins above could transduce into Jurkat-T cell efficiently.CCK-8 assay showed that PTD-hFOXP3 fusion protein could inhibit the proliferation of T cells.Conclusion:PTD-hFOXP3 and PTD-eGFP-hFOXP3 fusion proteins have been expressed successfully and will be a much help to better study the biological function of FOXP3 in Treg and elucidate the mechanism of immune suppression.