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崔中利, Zhong-Li Cui∗, Xiao-Zhou Zhang, Zhong-Hui Zhang & Shun-Peng Li
Biotechnology Letters 26: 1115-1118, 2004.,-0001,():
-1年11月30日
A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.
Bacillus subtilis,, gene expression,, genomic promoter library,, methyl parathion hydrolase,, promotertrapping vector
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【期刊论文】High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800
崔中利, Xiao-Zhou Zhang, Zhong-Li Cui, Qing Hong, and Shun-Peng Li*
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2005, p. 4101-4103,-0001,():
-1年11月30日
The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.
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【期刊论文】Expression, puriWcation, and characterization of a novel methyl parathion hydrolase
崔中利, Guoping Fu, a, b Zhongli Cui, a Tingting Huang, a and Shunpeng Lia, *
Protein Expression and PuriWcation 36(2004)170-176,-0001,():
-1年11月30日
The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identiWed. To further conWrm 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 puriWed to homogeneity by metal-aYnity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the puriWed recombinant MPH indicated that a signal peptide of the Wrst 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the puriWed 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 Wltration of the puriWed mature recombinant MPH revealed that the MPH was a monomer.
Methyl parathion hydrolase, Signal peptide
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崔中利, CUI ZHONGLI, LI SHUNPENG, * AND FU GUOPING
APPLIED AND ENVIRONMENTAL MICROBILOGY, Oct. 2001, p.4922-4925,-0001,():
-1年11月30日
A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp. strain M6 was able to hydrolyze methyl parathion to p-nitrophenol. A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the mpd gene was determined. The gene could be effectively expressed in Esherichia coli.
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崔中利, Ruifu Zhang, Zhongli Cui, Xiaozhou Zhang, Jiandong Jiang, Ji-Dong Gu & Shunpeng Li, *
Biodegradation (2006)17: 465-472,-0001,():
-1年11月30日
Seven organophosphorus pesticide-degrading bacteria harboring the methyl parathion degrading (mpd) gene were isolated from a methyl parathion contaminated site. In this study, the 4.7 kb mpd gene cluster, conserved in all seven bacteria capable of degrading methyl parathion, was cloned and further analysis revealed that this cluster contained five ORFs and the mpd gene was associated with a mobile element, IS6100. In addition to mpd gene ORF and tnpA ORF, three other ORFs showed high homology to the permease component of ABC-type transport system, the general secretion pathway protein B, and the RNA polymerase sigma 70 factor, respectively. The mpd genes of these 7 strains were subcloned and expressed in E. coli, SDS-PAGE and zymogram analysis showed that two expression products of mpd genes in E. coli were found, but the one without signal peptide showed the hydrolytic activities. Our evidences collectively suggest that mpd gene cluster may be disseminated through horizontal gene transfer based on phylogenetic analysis of the cluster and their host bacterial strains, and comparisons of GC content of the cluster and respective host’s chromosome.
horizontal gene transfer,, organophosphorus hydrolase gene cluster,, signal peptide,, degradation
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