胰岛素原基因的高效分泌表达系统——甲醇酵母Pichia pastoris

Highly secretory expression system for proinsulin gene:Methylotrophic yeast Pichia pastoris

为了提高重组人胰岛素的表达效率,使用了近年发展起来的真核表达系统-甲醇酵母Pichia pastoris. Pichia pastoris以甲醇为其惟一碳源,而乙醇氧化酶基因的启动子是强启动子,可用来调控异源蛋白的表达.该系统具有操作技术简单,可进行高水平的胞内或胞外表达,及对表达产物进行翻译后修饰与加工等显著优点.将含有小C肽或不含有C肽的人胰岛素原类似物基因整合到酵母基因组上来发酵生产人胰岛素原,在表达量上,每升发酵液中可以得到胰岛素原250～300mg,分泌水平约占总蛋白量的20%,重组人胰岛素具有与猪胰岛素相同的受体结合能力和生物活性.

To obtain the high-level expression of human insulin, the methylotropic yeast Pichia pastoris was used as expression system. Pichia pastoris uses methanol as a sole carbon source. Alcohol oxidase（AOX） promoter as one of the strongest promoters can regulate the expression of heterologous proteins. The system has many advantages： （1）the techniques needed for the molecular genetic manipulation of Pichia pastoris are simplier ； （2）Pichia pastoris can produce foreign proteins at high levels, either intracellularly or extracellularly； （3）the proteins can be performed many eukaryotic posttranslational modifications. The fused“mini-C” or “non-C” human proinsulin analog was cloned onto yeast chromosome, and under induction by methanol, the expressed proinsulin was secreted into medium at the level of 250～300 mg in 1 L fermentation culture. The expression level accounted for 20％ of total bacterial proteins. The recombinant human insulin has the same receptor binding capacity and biology activity with pig insulin.