Expression, purification, and characterization of a novel methyl parathion hydrolase

Protein Expression and Purification
Guoping FuShunpeng Li

Abstract

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.

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Citations

Jul 8, 2010·Applied Microbiology and Biotechnology·Xiao-yu ChuYun-liu Fan
Dec 31, 2011·Applied Microbiology and Biotechnology·Jijian YangChuanling Qiao
Jul 8, 2005·Applied and Environmental Microbiology·Xiao-Zhou ZhangShun-Peng Li
Apr 7, 2010·Journal of Bacteriology·Guangli WangJiandong Jiang
Feb 3, 2016·Environmental Science and Pollution Research International·Pauline JacquetEric Chabrière
Mar 8, 2011·Biotechnology Journal·Dau Hung AnhWerasak Surareungchai
Jan 30, 2021·Biotechnology Letters·Witsanu RapichaiJesdawan Wichitwechkarn

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