拟南芥细胞异三聚体G蛋白的分离纯化

Separation and purification of G protein from Arabidopsis thaliana

以拟南芥哥伦比亚Columbia（Col—O）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。

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 7 subunits. The purification method of G protein will facilitate further function investigation of G protein in mutant-type cell of A. thaliana.