毕赤酵母表达Neuritin蛋白的纯化及鉴定

Purification and Identification of Neuritin Protein in Pichia Pastoris

为研究重组Neuritin的毕赤酵母表达蛋白的纯化方法及鉴定。应用甲醇诱导GSll5/pPIC9K-Neuritin毕赤酵母工程菌株表达Neuritin蛋白,采用硫酸铵盐析、His-Trap Crud亲和层析等方法,对该表达产物进行纯化。纯化后的蛋白进行SDS-PAGE、western-blot和生物质谱鉴定。结果显示,SDS-PAGE分析显示经甲醇诱导后表达的蛋白获得了与Neuritin分子量理论值相符的条带,western-blot分析它能够与抗表位抗体特异反应,进一步生物质谱分析证实该纯化重组蛋白为Neuritin重组蛋白。由此可知,本研究建立了一种有效的纯化方法,获得高纯度的Neuritin重组蛋白,为大量制备Neuritin纯化蛋白,开展其功能和机制研究奠定基础。

To purify and identify recombinant Neuritin fusion protein in pichia pa storis.GSll5/pPIC9K-Neuritin Pichia pastoris was inducted by methanol to express Neuritin protein,which further purified by Amonium sulfate salting out,His-Trap Crud column affinity chromatography,then identified by SDS-PAGE,Western blot and mass spectrum.The results showed that induced by methanol,pichia pastoris expressed the right molecular weight protein.Western Blot test showed the purified protein had immune reaction with monoclonal specific antibody.Further biological mass spectrometry analysis confirmed that the purified recombinant proteins was Neuritin recombinant protein.The above purification method was a practical and effective way to obtain the high purity Neuritin recombinant protein and to prepare a mass production of Neuritin purified protein.It also laid the foundation for the further study of its function and mechanism.