Purification and biochemical characterization of a novel protein phosphatase-1 binding protein ,FLJ00215.

• Main Authors: Da-Xiang Gong, 龔大翔
• Other Authors: Hsien-Bin Huang
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/74720933008281035371

碩士 === 國立中正大學 === 分子生物研究所 === 96 === Protein phosphatase-1 is one of the major serine/threonine protein phosphatases in eucaryotiv cells. The catalytic subunit of protein phosphatase-1 (PP1) presents in cells as holoenzymes through association with a binding protein that targets the enzyme to specific subcellular compartments and to regulate its substrate function, including carbohydrate metabolism, protein synthesis, muscle contraction, transcription, cell cycle and neuronal signaling. In our lab, we have conducted the search for the new PP1-binding proteins of heart cells by using a yeast two-hybrid system and identified seven candidate proteins. One of them is encoded by FLJ00215. GST pull-down assay and co-immunoprecipitation have demonstrated that the translated product of FLJ00215 is associated with PP1 and targets the enzyme to Endoplasmic Reticulum. To better understand the biochemical functions of FLJ00215, I have prepared the recombinant thioredoxin-FLJ00215 fusion protein (Trx-FLJ00215) from E. coli expression system by using Ni+2-, Mono-Q Sepharose and size-exclusion chromatographies. SDS-PAGE analysis indicated the homogeneity of the purified Trx-FLJ00215 is more than 85%.We found that Trx-FLJ00215 can inhibit PP1 and PP2A activities with the IC50 of 10±3 nM and 187±9 nM, respetively. The purified Trx-FLJ00215 protein will be used for antibody preparation.