Study On Expression In Pichia Pastoris, Purification And Activity Assay Of The Fusin Protein Of Human Arginase And Humanalbumin

Objective: To develop an effectually secrete expression in Pichia pastoris and purification system of the fusin protein of human arginase and Humanalbumin (ARG-HSA), and to test the activity of ARG-HSA. Methods: Human arginase and Humanalbumin gene in the correct reading frame inserted into Pichia expression vector (pPIC9) after signal peptide, recombinant Pichia expression plasmid pPIC-Arg was transformed it into the host strain GS115. MTT assay was used to measure percent viability. Results: An recombinant expression plasmid pPIC-Hsa-Arg was generated successfully, and was identified by DNA sequencing; The recombinant protein was expressed in GS115 with high level, the recombinant arginase gene was expressed highly and secrete effectually in Pichia pastoris, and the expressed product had the normal bioactivity. The target protein can be released into the media, the expression of protein in the supernatants accounted for more than 90%. AfterUltrafiltration and gel filtration, the purity of recombinant Arg reached 95%, and the specific activity is about 310 IU/mg. All cell lines demonstrated decreased viability as concentrations of fusin protein of human arginase and Humanalbumin (ARG-HSA) increased. Conclusions: A set of protocols for high efficient the Pichia expression and purification has been established, which is simple, efficient and applicable. Fusin protein ARG-HSA was cytotoxic to melanoma cells, human laryngeal cancer cell and human hepatocellular carcinoma cell in vitro.