Translocation of recombinant protein via the Tat pathway in Escherichia coli
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. Howe...
| Main Authors: | , |
|---|---|
| Other Authors: | |
| Format: | Others |
| Language: | zh-TW |
| Published: |
2007
|
| Online Access: | http://ndltd.ncl.edu.tw/handle/36522683336972870141 |
| Summary: | 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.
|
|---|