Expression, purification, and characterization of a novel methyl parathion hydrolase.
The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.
Fu
Guoping
G
Key Lab of Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, 1 Weigang, Nanjing, Jiangsu 210095, People's Republic of China.
Cui
Zhongli
Z
Huang
Tingting
T
Li
Shunpeng
S
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008743:Methyl Parathion / Q000737:chemistry*; D016366:Open Reading Frames / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016981:Plesiomonas / Q000201:enzymology* / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D008743:Methyl Parathion; D006867:Hydrolases; D010744:Phosphoric Monoester Hydrolases