拟南芥细胞异三聚体G蛋白的分离纯化

以拟南芥哥伦比亚Columbia(Col-0)野生型悬浮培养细胞为材料,采用超声波破碎、匀浆、离心、40%~60%饱和度硫酸铵分步沉淀、Sephadex G-25脱盐、DEAE-Sepharose Fast Flow离子交换、Sephadex G-200凝胶过滤,最后经过Sepharose CL-6B得到纯化的目的蛋白,蛋白收率为0.097%。纯化的蛋白质经非变性聚丙烯酰胺凝胶电泳鉴定显示为一条带,经Western blotting证实为G蛋白。把经Native-PAGE鉴定的蛋白质的条带回收,进行SDS-PAGE显示有3条带。一条是Gα亚基,其分子量为60kDa左右;另外2条带分子量为45kDa和35kDa,可能是β、γ亚基,初步证实拟南芥中存在异三聚体G蛋白。G蛋白提取方法的建立为在基因突变型拟南芥中G蛋白功能的研究奠定基础。

Separation and purification of G protein from Arabidopsis thaliana

G protein was purified from the suspended cultured wide-type cell of Arabidopsis thaliana (Col-0) by ultrasonic fragmentation,homogenization,centrifugation,40%～60% saturation Ammonium sulphate sedimentation,gel filtration (Sephadex G-25,Sephadex G-200,Sepharose CL-6B) and DEAE-Sepharose Fast Flow ion exchange chromatographer. The result identified by non-denatured PAGE showed one band in the gel and Western blotting analysis confirmed that the protein was G protein. The target protein after Native-PAGE was collected,and it displayed three bands after SDS-PAGE. The first band was α subunit,and its MW was about 60kDa. The second and third bands which were 45kDa and 35 kDa,were presumed to be the β and γ subunits. The purification method of G protein will facilitate further function investigation of G protein in mutant-type cell of A.thaliana.