Purification And Liposome-coation Of Recombinant T4Endonuclease V (T4N5)

Purpose: To prepare T4endonuclease V (T4N5) by fusion expression in E.coli and thento coat it with liposomeMethods: Constructing the recombinant expression vector pET-32a-thrombin-T4N5through total gene synthesis the T4N5gene sequences. These vectors were transformedinto Origami DE3. The synthesized plasmid was sequenced and then protein expression isvalidated. The high-purity T4N5was separated by nickel affinity chromatography andC18reverse-phase chromatography from the fusion, which was cleavaged by thethrombin. The activity of T4N5, coated by liposomes, was detemirmined by in vitro andanimal experiments.Result: Result: Recombinant vectors pET-32a-thrombin-T4N5were successfullyconstructed The recombinant plasmids were transformed into Origami DE3. Induced withIPTG, the expression level of fusion protein was over20%of total bacterial protein. Thedigestion efficiency is not less than90%. The efficiency of thrombin was more than80%.Molecular weight and amino acid sequence of prepared T4N5was consistent withtheoretical value，and its purity was over90%. The UV resistance activity results showedit could repair UV-induced DNA damage.