Design and structure determination of a thermostable multimeric DNA binding protein using Sac7d as a template

• Main Authors: Wu Sz-Wei, 吳思緯
• Other Authors: Andrew H. J. Wang
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/09198540338279970987

碩士 === 國立臺灣大學 === 生化科學研究所 === 91 === The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Sac7d is extremely stable to heat, acid and chemical agents. This protein binds DNA without any particular sequence preference. We propose that multimeric Sac7d would show different DNA binding properties. In order to form dimeric Sac7d, a helical peptide was fused after the C-terminal end of Sac7d. Two or more monomers theoretically will interact with each other via hydrophobic force, as in leucine zipper, to form a dimer. Large amount of recombinant Sac7d was obtained by expression in E. Coli and purification by heating and ion-exchange chromatography. The physical and chemical properties of Sac7d were verified by gel-filtration, chemical cross-linking with SDS-PAGE, SPR, and CD. The tertiary structure was determined by X-ray crystallography. This model works as a successful template that endows protein new function without losing original properties. Furthermore, a thermo-stable enzyme that targets DNA, such as Tag DNA polymerase using in PCR, deriving from Sac7d might be practicable, or fusing a transmembrane domain to Sac7d which inlays in micelles works as a highly stable DNA carrier used in gene therapy.