Application of zinc finger protein-conjugated sensor chip for the detection of zinc ions
| Autor: | Chun-Chang Lin, 林鈞章 |
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| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 95 Metal-responsive transcription factor 1 (MTF-1) is a transcription factor which regulates the gene expression of several metal inducible genes. The N-terminus of MTF-1 is consist of six zinc fingers, and the C-terminus contains three domains associated with transcriptional activation. The zinc finger domains of MTF-1 are able to sense the concentration change of Zn(Ⅱ) and bind to the metal response elements (MREs). In this study, we attempt to purify the zinc finger domains of MTF-1, and try to establish a biomolecule-based biosensor system which is specific for Zn(Ⅱ) detection through the combination of zinc finger protein-conjugated sensor chip and surface plasmon resonance technology. We expressed the glutathione-S-transferase and zinc finger protein fusion protein (GST-6ZF) in E. coli. This fusion protein was initially purified with glutathione beads, followed by gel filtration chromatography. The purified GST-6ZF was immobilized onto a carboxy- methylated dextran matrix (CM5 chip). We evaluted the interaction at different pH values between the zinc finger sensor chip and six metal ions, including Zn(Ⅱ), Ca(Ⅱ), Cd(Ⅱ), Ni(Ⅱ), Co(Ⅱ), and Mg(Ⅱ). It is found that the interaction between zinc finger sensor chip and Zn(Ⅱ) is much stronger than other metal ions at pH 4. The sensitivity of the zinc finger sensor chip to Zn(Ⅱ) is also evaluated. The sensor chip detected the zinc concentration is low as 1μM. Our results show the possibility of using MTF-1 to develop the detection system for bioavailable zinc ions. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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