人精氨酸酶在毕赤酵母的高效表达、纯化和活性研究

目的：人精氨酸酶（Arginase, Arg）的基因arg在毕赤酵母高效分泌表达，建立相应纯化工艺路线，研究重组人精氨酸酶的活性。方法：将人精氨酸酶基因arg按正确的阅读框架插入到毕赤酵母表达载体pPIC9α信号肽基因后，构建得到重组毕赤酵母表达质粒。转化毕赤酵母GS115筛选高表达菌株。结果：成功构建了酵母表达载体pPIC-Arg，转化毕赤酵母GS115后筛选到分泌表达目的蛋白Arg的菌株，目标蛋白可以分泌到培养基中。经过膜过滤和凝胶过滤层析对培养基上清进行纯化，即可获得纯度达到95%的活性产物。活性测定表明，纯化的Arg比活性为310 IU/mg。结论：成功构建了Arg的毕赤酵母高效表达菌种，建立了目标物质的分离纯化工艺。

Study on Expression in Pichia pastoris, Purification and Activity of Human Arginase

Objective To develop an effectually secrete expression in Pichia pastoris and purification system of recombinant human Arginase(Arg), and to test the activity of recombinant Arg. Methods Human arginase 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. Results An recombinant expression plasmid pPIC-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 80%. AfterUltrafiltration and gel filtration, the purity of recombinant Arg reached 95%, and the specific activity is about 310 IU/mg. Conclusions A set of protocols for high efficient the Pichia expression and purification has been established, which is simple, efficient and applicable.