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Purification and characterization of recombinant human liver prolidase expressed in Saccharomyces cerevisiae

  • Toxicokinetics and Metabolism
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Abstract

The recombinant human liver prolidase (rh-prolidase, EC 3.4.13.9) from the lysate supernatant of engineering yeast Saccharomyces cerevisiae was purified in two steps employing anion-exchange gradient chromatography (DEAE-Sepharose fast flow) and gel filtration chromatography (Sephacryl S-200 high resolution). The purified recombinant protein furnished a single band with a molecular weight of 56 kD. Intensity scanning of the SDS-PAGE gel revealed that the prolidase accounted for more than 90% of total protein. The optimum pH of the catalytic reaction was 8.0. The enzyme was stimulated by Mn2+, but strongly inhibited by Cu2+ and Zn2+. The rh-prolidase expressed in S. cerevisiae had both dipeptidase and organophosphorus acid anhydrolase activity. It catalyzed the hydrolysis of soman and the dipeptide Gly–Pro. In a detoxification test in vitro, purified rh-prolidase was remarkably efficient at eliminating the toxicity of a lethal dose of soman, with the result that mice survived injection of such a dose.

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Authors and Affiliations

  1. Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, Tai-Ping Road 27, Beijing, 100850, China

    Shu-Hao Wang & Man-Ji Sun

  2. Institute of Rice, Sichuan Agricultural University, Wenjiang, 611130, Chengdu, Sichuan, China

    Qing-Wen Zhi

Authors
  1. Shu-Hao Wang
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  2. Qing-Wen Zhi
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  3. Man-Ji Sun
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Correspondence to Man-Ji Sun.

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Wang, SH., Zhi, QW. & Sun, MJ. Purification and characterization of recombinant human liver prolidase expressed in Saccharomyces cerevisiae. Arch Toxicol 79, 253–259 (2005). https://doi.org/10.1007/s00204-004-0634-4

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  • DOI: https://doi.org/10.1007/s00204-004-0634-4

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