Engineering a thermo-stable protein with two DNA-binding domains
| Autor: | Jr-Ming Yang, 楊之茗 |
|---|---|
| Rok vydání: | 2003 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 91 The acid- and thermo-stable Sac7d is a small, non-specific DNA- binding protein of the hyperthermophile Archaea Sulfolobus acidocaldarius. In this thesis, Sac7d is employed as a functional unit in the design of a thermo-stable protein containing two putative DNA-binding domains. An expression vector carrying a single mutation of the C-terminal lysine to cysteine is constructed. Dimeric Sac7d is obtained by linking two mutant Sac7d molecules through a C-terminal disulfide bond. Thermal stability and DNA-binding ability of dimeric Sac7d are assessed and compared to those of wild type Sac7d by gel retardation assay, circular dichroism spectroscopy, and x-ray crystallography. Dimeric Sac7d is shown to be equally thermo-stable as wild type; however, the interaction of dimer with various DNA sequences diverges from that of wild type, suggesting a different DNA-binding mode for dimeric Sac7d. The possible application of the mutant Sac7d protein with a reactive thiol group is also discussed. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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