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.
Dept. of Biochemistry, S K University, Anantapur 515 003, India.
Sita
S
Somara
Bramanandam
B
Manavathi
Christoph C
CC
Tebbe
Dayananda
D
Siddavatam
D020816:Amino Acid Motifs; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005417:Flavobacterium / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry*; D006867:Hydrolases / Q000737:chemistry*; D007477:Ions; D008970:Molecular Weight; D010278:Parathion / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014158:Transcription, Genetic; D015032:Zinc / Q000737:chemistry
D007477:Ions; D011994:Recombinant Proteins; D006639:Histidine; D010278:Parathion; D006867:Hydrolases; C574976:parathion hydrolase, Flavobacterium balustinum; D010744:Phosphoric Monoester Hydrolases; D015032:Zinc