Over-expression of parathion hydrolase of Flavobacterium balustinum in E. coli: purification and characterization of His-tagged parathion hydrolase.

The organophosphorus pesticide degrading (opd) 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 E. coli JM105 and E. coli BL21 respectively. On induction the E. coli cells containing pNH2 produced high levels of parathion hydrolase. A 60 kD fusion protein was produced in E. coli 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 His-tag was removed by digesting it with thrombin. The resulting protein folded properly in presence of Zn2+ ions, and showed parathion hydrolase activity.