Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis

Abstract

Using Sso7d from Sulfolobus solfataricus as the DNA binding protein fused to Taq DNA polymerase at its amino terminus, we report the hyper-expression and a novel purification methodology of Sso7d-Taq polymerase (S-Taq) using aqueous two-phase extraction system followed by Ni-affinity chromatography. The utility of such a fusion enzyme in carrying out PCR of human genes from whole blood directly and in detecting hepatitis B virus from clinical samples is demonstrated in this article. We present data on the enhanced thermo-stability of S-Taq DNA polymerase over Taq DNA polymerase and also provide evidence of its higher stability with detergents in comparison to Taq polymerase. The purified S-Taq protein showed acceptable limits of host genomic DNA levels without the use of DNases and other DNA precipitating agents and shows promising potential for use in PCR based diagnostics, in-situ PCR’s and forensic science. Keywords: Aqueous two phase extraction system, Ni-NTA chromatography, Taq polymerase, Sso7d fusion proteins