Chain The Nature And Function Of The Mold The Phage ��c31 Integrase Enzyme And Its Application In Mammalian Cells

Despite some success in the field of gene therapy and transgenic organisms,the research community is still facing an old problem,namely,adopting gene transfer approaches deliver a gene to target site-specific mammalian chromosome integration without adverse events and mutagenesis caused by random insertion.To minimize the potential disruption of genomic integrity,a target gene transfer should be inserted at a specific site proven to be a safe genomic location.Recently,Streptomyces phage F C31 integrase was reported to be able to mediate exogenous DNA containing wild att sequences site-specific integration into pseu-att sites on mammalian cellular genome.It is possible that the F C31 integrase system has great potential for a tool of gene manipulation and gene therapy.There have been several gene transfer approaches to deliver corrective genetic materials into target cells,some reported incident have been raising concerns about the safety of gene delivery viral vectors.Non-viral gene delivery approaches,such as naked DNA vector was advancing to gene delivery with poor transfection efficiency in some cell types and in vivo,and the potential for random integrating into the host genome resulting in continued expression.A new protein transduction technique was developing in several past years,that can deliver a biologically active enzyme to all cells and tissue of mice,thereby,overcoming limitations of DNA transfection and viral infection.It suggests that this new technique can be used as a tool of delivery F C31 integrase to mammalian cells for the sake of efficiency and safe.The catalytic activity and biological characterization of Streptomyces phage F C31 integrase was be studied by our established an in vitro linear intramolecular assay system and successfully expressed a C-terminal 6His-taggedφC31 integrase fusion protein in Escherichia coli BL21(DE3).φC31 integrase purified in a single Ni-NTA affinity chromatographic step to a specific activity of 23.8U/mg protein at optimum pH 8.5 and 20℃.The partial purified enzyme was highly active in the pH and temperature ranges of pH 6.5 to pH 11,and temperature from 9℃to 37℃,respectively.Its catalytic activity was enhanced in the presence of K⁺,corresponding to the results of the purified protein contained more K⁺ other than Ca²⁺,Mg²⁺ or Mn²⁺ detected by the inductively coupled plasma mass spectroscopy(ICP-mass).The K_m and V_max of the recombinantφC31 integrase were 6.261 nmol/L and 0.8621 nM^-1 min^-1,respectively.The stability of the purified protein showed that the protein second structural changes are sensitive to the conditions of pH,temperature,metal ions and DNA substrates as well,investigated by circular dichroism(CD) and Fourier-transform infrared(FF-IR) spectroscopy.All results indicated thatφC31 integrase was a member of serine recombinase family,but not characteristically and mechanistically,analogous toλ.recombinases,such as Cre recombinase.The catalytic and conformational characterization ofφC31 integrase will provide a base to catch on emzymic structure and function relationship of the large serine recombinase subgroupIn order to new application ofφC31 integrase protein transduction,fusion 11 amino acids peptide of HIV-1 Tat protein toφC31 integrase was expressed and purified,which can enter into mammalian cells and perform recombination function in the manners of dose-dependency in our established mammalian cellular quantitative assay system.The results were demonstrated using a fluorescence-activated cell sorter analytical flow cytometer and indirect immunofluorescence.The study of relationship betweenφC31 integrase activity and its sub-cellular localization in mammalian cells,indicates that nuclear translocation is a limiting factor to F C31-mediated recombination at genome level in mammalian cells.A nuclear localization signal(NLS) of SV40 large T antigen modified C-terminalφC31 integrase was designed to transferφC31 integrase into cells by protein transduction and DNA transfection.Consequently,a TAT-F C3-NLS protein transduction was used to mediate human coagulating factorâ…¨(hFâ…¨) gene site-specific integration into mammalian genome,those isolated and expanded cell lines can express hFâ…¨and have about 50%integration in cellular genomic psiA sites. Transient TAT-F C31-NLS displayed in transduced cells by immunoblotting analysis and had no influence on the cell cycle and early apoptosis in human embryonic kidney 293-cell and Monkey kidney COS-1 cell.TAT-F C31-NLS turned out to have efficient transducible ability and high recombination function in mammalian cell,and can be produced readily from bacteria expression on a large scale and purified rapidly by a single affinity chromatographic step under native conditions.We expect that TAT-F C31-NLS protein transduction can apply to serve as a useful tool for genetic manipulation and gene therapy.In conclusion,the results of catalytic characterization of Streptomyces phage F C31 integrase in vitro provide a base for its application in gene manipulation and gene therapy. We introduce F C31 integrase protein transduction into mammalian cells supplying the catalytic F C31 integrase with non-viral vector system in the form of DNA,which has low transfection efficiency in particular cell types and the potential for integrating into the host genome resulting in continued expression.Internalized F C31 integrase protein mediated hFâ…¨gene site-specific integration in mammalian genome represents an effective site-specific integration system for cells and may be of value in gene therapy and other chromosome engineering strategies.