Molecular Cloning, Expression, and Functional Characterization of a Cystatin from Pineapple Stem

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A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (<I>Ananas comosus</I>) stem. This clone was constructed in a fusion vector and was easily over-expressed in <I>Escherichia coli</I>; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity <I>via</I> a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable <I>K</I><SUB>i</SUB> values of 1.18×10<SUP>−10</SUP> <small>M</small> and 9.53×10<SUP>−11</SUP> <small>M</small>, respectively. The recombinant cystatins were found to be thermally stable up to 60 °C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.

収録刊行物

  • Bioscience, biotechnology, and biochemistry

    Bioscience, biotechnology, and biochemistry 68(8), 1681-1689, 2004-08-23

    公益社団法人 日本農芸化学会

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各種コード

  • NII論文ID(NAID)
    10013520710
  • NII書誌ID(NCID)
    AA10824164
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    09168451
  • NDL 記事登録ID
    7066896
  • NDL 雑誌分類
    ZR7(科学技術--農林水産--農産) // ZR2(科学技術--生物学--生化学) // ZP1(科学技術--化学・化学工業)
  • NDL 請求記号
    Z53-G223
  • データ提供元
    CJP書誌  NDL  J-STAGE 
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