Overexpression, purification, characterization and crystallization of the DNA binding proteins from Sulfolobus solfataricus ATCC 35092

• Main Author: 吳淑蓉
• Other Authors: 李其融
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/87370525187807092891

碩士 === 明新科技大學 === 化學工程與材料科技系 === 99 === Sulfolobus solfataricus is a thermoacidophilic archaeon which grows optimally at approximately 75~85C and pH 2~4. It contains a number of small basic DNA-binding proteins ranging in molecular weight from 7 to 10 KDa (e.g.Sso10b, Sso7d and Sso7c4). It is commonly believed that members of the Sso7d protein family and other related proteins play an architectural role in packaging and stabilizing genomic DNA at the high growth temperatures of these hyperthermophiles . The gene encoding DNA binding protein (i.e. Sso7c4) was amplified by PCR from the genomic DNA of Sulfolobus solfataricus P2. The gene was cloned into the pET-29a vector (Novagen) with NdeI and BamHI sites. Large amount of the recombinant protein was over-expressed in E. coli. (DE3) codenplus-RIL and purified with high purity by heating, cation-exchange chromatograpy and dialysis. The spectroscopic methods such as UV-VIS, fluorescence and CD have been used to study variety properties of these DNA binding proteins in solution. We also tried to obtain the crystal of the DNA binding protein only or in complex with the dsDNA by vapor diffusion method and determine the crystal structure by X-ray diffraction.