Over-expression of parathion hydrolase of <i>Flavobacterium balustinum </i>in <i>E. coli:</i> Purification and characterization of His-tagged parathion hydrolase

Abstract

The organophosphorus pesticide degrading (<i>opd</i>)<i> </i>gene was cloned downstream to the transcriptional and translational signals of expression vectors pTrc99A and pET32A. The resulting recombinant expression plasmids pNH2 and pHH2 were introduced into <i>E. coli </i>JM105 and <i>E. coli </i>BL21 respectively. On induction the <i>E. coli </i>cells containing pNH2 produced high levels of parathion hydrolase. A 60 kD fusion protein was produced in <i>E. coli </i>cultures containing recombinant plasmid pHH2. The molecular mass of the fusion protein coincided with the molecular mass of 40 kD parathion hydrolase and 20 kD N-terminal His tag encoded by the vector. Further, the fusion protein was purified using Ni-column and the N-terminal Histag was removed by digesting it with thrombin. The resulting protein folded properly in presence of Zn²⁺ ions, and showed parathion hydrolase activity.