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Giới thiệu nội dung

The XthA Gene Product of Archaeoglobus Fulgidus Is an Unspecific DNase

Tác giả: Mandy Miertzschke and Thomas Greiner-Stöffele

Lĩnh vực: Protein Engineering, Biochemistry

Nội dung tài liệu: This study investigates the expression and characterization of the xthA gene product from Archaeoglobus fulgidus, a hyperthermophilic archaeon. The research aimed to determine if this enzyme is homologous to exonuclease III from Escherichia coli. Through sequence similarity searches and experimental analysis, the study found that the xthA gene product exhibits strong DNA binding properties and nicking activities on double-stranded DNA, behaving as an unspecific DNase. Unlike exonuclease III, it can degrade supercoiled plasmids and shows no preference for specific DNA termini. The enzyme’s activity is inhibited by EDTA and is only weakly active when magnesium ions are replaced by calcium ions. The research also explored the enzyme’s optimal temperature and pH, its dependence on divalent ions, and its substrate specificity, providing insights into its potential catalytic mechanism.

Mục lục chi tiết:

  • Materials and methods
  • Bacterial strains and plasmids
  • Media, oligonucleotides and enzymes
  • Cloning of the xthA gene of A. fulgidus
  • Expression of the xthA gene product of A. fulgidus
  • Purification
  • Characterization
  • Temperature profile
  • pH profile
  • Ion dependencies
  • Time profiles Hepes, Mg2+ and Ca2+
  • Substrate specificity profile
  • Nicking activity
  • Discussion
  • References