Translocation of recombinant protein via the Tat pathway in Escherichia coli

• Main Authors: Jhih-Sian Ye, 葉志賢
• Other Authors: 林松池
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/36522683336972870141

碩士 === 中興大學 === 化學工程學系所 === 95 === Gram-negative bacteria such as Escherichia coli have multiple pathways for exporting secretory proteins. The Twin-arginine translocation (Tat) pathway was recently found to be a novel system which is capable of translocating folded proteins into the periplasm. However, the translocation efficiency of the Tat pathway is not as high as that of the Sec pathway. It has been previously shown that the co-expression of TorD is capable of enhancing the translocation of green fluorescence protein (GFP) with TorA signal peptide. One of the objectives of this study is to identify the optimal stoichiometric ratio between GFP and TorD for protein secretion. To this end, three recombinant E. coli strains harboring plasmids encoding GFP fusion and TorD were constructed. Periplasmic and cytoplasmic fractions of the cells were fractionated by sucrose gradient centrifugation. Western blotting analysis and fluorescence spectroscopy were used to evaluate the efficiency of protein translocation.