A Study on Intracellular Expresstion of Human Angiostatin K1-3 in Pichia Pastoris

Objective:To construct a recombinant plasmid PHIL-D2/K1-3 for the expression of human AngiostatinK1-3 in Pichia pastoris.Methods :The gene of human AngiostatinK1-3 was amplified from the embryo liver tissue using RT-PCR and then cloned into PHIL-D2 vector.After being linearized,the recombinant plasmid was transformed into Pichia pastoris GS115.The positive transformant was identified by PCR and Southern blotting and then induced by methanol.The expressed products was analyzed by SDS-PAGE(12%) and Western blot.After being purified by lysine/Sepharose-4B affinity chromatography,the recombinant protein concentration was determined with Lowry's method and its antiangiogenic activity was assayed as well.Results: The recombinant protein was about 30 KD.and the level of expression was 6.8mg/L.In the activity assay,the recombinant protein could strongly inhibit the proliferation of HUVEC.Conclution: The recombinant human AngiostatinK1-3 is successfully expressed in Pichia pastoris GS115.