毕赤酵母表达人Neuritin蛋白的条件优化、纯化及鉴定

Purification,Identification and Optimized Expression of Recombinant Human Neuritin in Pichia Pastoris

对毕赤酵母表达重组Neuritin蛋白的产物进行了纯化和鉴定,并进行了细胞毒性的量效关系的研究,为进一步应用于动物模型的实验研究奠定基础。应用甲醇诱导毕赤酵母工程菌株GS115/pPIC9K-rhNeuritin表达Neuritin蛋白,对甲醇诱导的浓度和时间进行了优化。表达产物经Ni-NAT纯化和透析浓缩,进行SDS-PAGE和Western-blot鉴定,将纯化产物用于体外培养的大鼠胸主动脉平滑肌细胞,对Neuritin蛋白与细胞存活的量效关系进行了研究。条件优化的结果显示,1%甲醇浓度诱导表达96h时,Neuritin蛋白表达量最大,可达0.48mg/mL。SDS-PAGE确定了表达产物分子量的大小,约为11kda;Western-blot证实表达产物为Neuritin;进一步的细胞毒性实验显示,纯化后的Neu-ritin蛋白在2.5μg/mL的浓度比较适合细胞生存。毕赤酵母高效分泌性表达了人Neuritin蛋白,经过镍柱亲和层析得到了有效的纯化,其发挥生物学作用的蛋白浓度为2.5μg/mL。

To purify and identify the human Neuritin expressed in Pichia pastoris under methanol induction and study the dose-effect relationship between Neuritin and cytotoxicity to lay the foundation for further animal model research. Applied methanol to induce pichia pastoris engineering strain to express Neuritin protein and optimize concentration and time of methanol induction. The expression product was identified by SDS-PAGE and western-blot after Ni-NAT purification and dialysis. The dose-effect relationship between purified Neuritin and cell survival were studied by the way of adding Neuritin into the medium of rat aortic smooth muscles ceils. After inducting 96h by 1% methnol concentration, Neuritin reached the maximum expression level. The quantitative determination was 0.48mg/mL. The results of Western-blot and cell culture showed the expression product had immunogenicity and biological activity. The optimal concentration of Neuritin was 2.5ug/mL for cell survival. The recombinant human Neuritin got efficient secretory expression in Pichia pastoris and hade certain biological activity after Ni-NAT efficient purification.