Study of affinity tag recombinant proteinexpression and purification system

• Main Authors: Fang-ying Chang, 張芳瑛
• Other Authors: 吳烘
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/93234439180372720228

碩士 === 南台科技大學 === 生物科技系 === 98 === Expression and purification of protein of interest is an essential step in study of the functional significance of protein. In genetic engineering, recombinant proteins containing affinity tags are often used to facilitate the purification process and have shown to increase yield and solubility.Lipid rafts are specialized microdomain of plasma membrane characterized and played an important role in vecicular trafficking and receptor-mediated signal transduction. Previous studies indicate the poteomic analysis of lipid rafts components in monocytic THP-1 cells, including several signaling molecules, such as Giα2、Giα3、Ral-A、Syntenin. Syntenin is an adaptor-like molecule containing two PDZ domains. Syntenin functions in a variety of biological processes such as activation of the transcription Sox4, migration and the regulation of membrane-cytoskeleton interactions, cell adhesion. Syntenin could be identified of interacting partners associate with syntenin-1 through direct interaction with its PDZ domains by yeast two-hybrid assays and immunoprecipitation assays. In contrast, affinity tag proteomics strategy are rarely used to investigate of syntenin functions.Fractalkine (CX3CL1) is a CX3C chemokine consisting of chemokine domain, mucin-like stalk, single transmembrane region, and a short intracellular C terminus. Fractalkine exhibits chemoattractant activity. It mediates its effects by binding and activating the CX3CR1 receptor.In lipid rafts investigation, discussed the changes of raft components in fractalkine-treated THP-1 cells. However, the functional significance of the mucin-like domain and the glycosylation modification in chemokine domain is still not clearly.The aim of this study is to express and purify of recombinant protein in order to investigate its biologic functions. To facilitate purification of recombinant protein, a variety of affinity tags were attached in the constructs. This study highlights a successful strategy using genetic engineering and protein purification technology in establishment of expression system for the study of functional significance of target protein.