GPD1酶在毕赤酵母中的表达及纯化

Expression and purification of glycerol-3-phosphate dehydrogenase 1(GPD1)in Pichia pastoris

目的构建可以分泌表达GPD1酶的毕赤酵母,并使用Profinia蛋白纯化系统获得纯度90%以上的GPD1酶,扩大GPD1酶的制备,以进一步研究其在肿瘤中的功能及作为抗肿瘤靶点的临床应用研究。方法提取人细胞的总RNA,逆转录成cDNA,以此为模板通过PCR扩增GPD1基因,PCR产物与表达载体pPICZα-C用EcoRⅠ和XbaⅠ双酶切,连接构建重组表达质粒pPICZα-C-gpd1;pPICZα-C-gpd1质粒电转至毕赤酵母X-33中,筛选阳性转化子,经甲醇诱导表达,采用固化金属亲和层析(IMAC)纯化分泌的目的蛋白。结果经SDS-PAGE鉴定,成功表达和纯化了GPD1酶,纯度达90%以上。结论本研究建立了毕赤酵母分泌表达GPD1酶的方法,可用于制备GPD1酶,为进一步的肿瘤机制研究和应用研究奠定基础。

Objective To construct a Pichia pastoris vector with expression of GPD1 enzyme and use profinia protein purification system to obtain GPD1 enzyme for further study the GPD1 function in cancer biology and clinical application as the anti-cancer target.Methods GPD1 gene was amplified by PCR using total RNA extracted from human cells and reverse transcribed into cDNA.The PCR product and vector pPICZα-C were digested by EcoRⅠand XbaⅠ,and used to construct the recombinant expression vector pPICZα-C-gpd1.The pPICZα-C-gpd1 was transformed into Pichia pastoris X-33 by electroporation.The positive colonies were selected and induced by methanol.Immobilized metal affinity chromatography(IMAC)was used to purify GPD1.Results GPD1 enzyme was successfully expressed and purified with a purity of more than 90%by SDS-PAGE identification.Conclusion This study establishes a method to express GPD1 enzyme in Pichia pastoris,which can be used to prepare GPD1 enzyme,and lays a foundation for further research on tumor mechanism and application.