乙酰氨基半乳糖转移酶14(GALNT14)在毕赤酵母中的表达纯化及活性鉴定

Expression,Purification and Activity Analysis of Human GALNT14 in Pichia Pastoris

将GALNT14全长编码区克隆到分泌型酵母表达载体pPIC9K中,构建重组载体pPIC9K-T14,经电转至毕赤酵母GS115中表达,使用G418筛选高表达重组菌,并对诱导条件进行优化,表达产物使用SDS-PAGE分析、Western blot鉴定、Sephadex G-100纯化、最后经HPLC检测活性。结果显示,在提供更好的供氧量条件下,用0.75%甲醇诱导可提高目的蛋白的表达量而降低杂蛋白的表达。培养上清液经SDS-PAGE检测显示目的蛋白分子量约64 kDa;Western blot结果显示在相应分子量处有一条特异性条带;利用Sephadex G-100成功分离纯化了目的蛋白;活性测定结果显示所获得的目的蛋白具有催化活性,为深入研究GALNT14的结构与功能奠定了基础。

The full-length cDNA of GALNT14 was cloned into the pPIC9K vector. The recombinant vector was transformed into Pichia pastoris GS115 using electroporation. The recombinant strains with high expression of GALNT14 were screened using G418. Different methanol concentration was applied to optimize the cultivation conditions. The expressed recombinant protein was detected by SDS-PAGE and Western blotting, then purified by Sephadex G-100. The activity of recombinant protein was analyzed with HPLC. The result showed that the protein production was increased with better oxygen supply in the cultivation and 0.75% methanol. The SDS-PAGE analysis indicated that the apparent molecular weight of target protein was about 64 kDa. Western blotting analysis suggested that it was human GALNTI4 protein. Enzyme activity assay glycosyhransferase activities, which provide a basis for the further study on GALNT14 showed the purified GALNT14 has the function and structure of the