Journal
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
Volume 176, Issue 7, Pages 1859-1869Publisher
SPRINGER
DOI: 10.1007/s12010-015-1683-2
Keywords
DNA ligase; DNA-binding domain; Long-range PCR; Heparin
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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 40-70 degrees C, and both were inactivated via heating at 95 degrees 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.
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