DNA-Binding Domain of DNA Ligase from the Thermophilic Archaeon Pyrococcus abyssi: Improving Long-Range PCR and Neutralization of Heparin’s Inhibitory Effect

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• 作者：Igor P. Oscorbin ; U. A. Boyarskikh…
• 关键词：DNA ligase ; DNA ; binding domain ; Long ; range PCR ; Heparin
• 刊名：Applied Biochemistry and Biotechnology
• 出版年：2015
• 出版时间：August 2015
• 年：2015
• 卷：176
• 期：7
• 页码：1859-1869
• 全文大小：658 KB
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• 作者单位：Igor P. Oscorbin (1) (2)
  U. A. Boyarskikh (1)
  A. I. Zakabunin (1)
  E. A. Khrapov (1)
  M. L. Filipenko (1)
  
  1. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russian Federation
  2. Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russian Federation
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Biochemistry
  
• 出版者：Humana Press Inc.
• ISSN：1559-0291

文摘

The DNA-binding domain of the DNA ligase from Pyrococcus abyssi (PabDBD) was mapped and cloned into two expression vectors. The resulting 6X His-tagged proteins, with a predicted molecular mass of approximately 30?kDa, were overexpressed, purified using Ni-NTA resin, and biochemically characterized. Both PabDBD derivatives bound to double-stranded DNA fragments at the temperature range of 40-0?°C, and both were inactivated via heating at 95?°C for 15?min. Complexes of the PabDBD variants with either double- and single-stranded DNA fragments were less stable than the native DNA ligase of P. abyssi. Inclusion of the C-terminally 6X His-tagged PabDBD in the reaction mixture during long-range polymerase chain reaction (PCR) increased the efficacy of amplification and eliminated the inhibitory effect of heparin.