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Author(s): |
Xueting Yang, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan, China, 430079
Yongze Yuan, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan, China, 430079
Meng Lou, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan, China, 430079
Ningning Zhang, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan, China, 430079
Junzhong Yang, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan, China, 430079
Hui Geng, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan, China, 430079
Li Xiong, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan, China, 430079
Deli Liu, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan, China, 430079 |
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Abstract: |
Methyl parathion hydrolase (MPD) is the key enzyme for methyl parathion degradation. MPD has been widely used to wipe off organophosphorus pesticides. Usually, recombinant MPD was expressed in E.coli BL21 cells predominantly as inclusion bodies with little active enzyme to collect. In this paper, we studied the denaturing of MPD inclusion bodies by urea as well as the protein renatured by diluting and dialyzing. We investigated the effect of urea concentration on the denature efficiency of MPD inclusion bodies and the certain factors affecting the efficiency of protein renaturation. These factors are including renature buffer components, pH, GSH/GSSG, and the protein concentration in the renature complex. The results suggested that the optimum urea concentration is 8 M for denature, and the optimum renature condition was 49? g/ml denature proteins diluted in Tris-HCl buffer (pH 8.5) plus 5 mM GSH, 0.5 mM GSSG and 1.5 M urea. Consequently, the renatured MPD reached the electrophoresis purity and its activity was 2.4 U/ml. The specific activity of the MPD reached at 48.8 U/mg, and the renature ratio of the recombinant MPD was 12.5% comparing with the activity of that expressed as soluble proteins.
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